U.S. patent number 6,071,922 [Application Number 09/044,558] was granted by the patent office on 2000-06-06 for synthesis, anti-human immunodeficiency virus, and anti-hepatitis b virus activities of 1,3-oxaselenolane nucleosides.
This patent grant is currently assigned to Emory University, The University of Georgia Research Foundation, Inc.. Invention is credited to Chung K. Chu, Jinfa Du, Raymond F. Schinazi.
United States Patent |
6,071,922 |
Schinazi , et al. |
June 6, 2000 |
Synthesis, anti-human immunodeficiency virus, and anti-hepatitis B
virus activities of 1,3-oxaselenolane nucleosides
Abstract
A method and composition for the treatment of HIV infection, HBV
infection, or abnormal cellular proliferation in humans and other
host animals is disclosed that includes the administration of an
effective amount of a 1,3-oxaselenolane nucleoside or a
pharmaceutically acceptable salt thereof, optionally in a
pharmaceutically acceptable carrier.
Inventors: |
Schinazi; Raymond F. (Decatur,
GA), Chu; Chung K. (Athens, GA), Du; Jinfa (Irvine,
CA) |
Assignee: |
Emory University (Atlanta,
GA)
The University of Georgia Research Foundation, Inc. (Athens,
GA)
|
Family
ID: |
21915638 |
Appl.
No.: |
09/044,558 |
Filed: |
March 19, 1998 |
Current U.S.
Class: |
514/274;
544/317 |
Current CPC
Class: |
A61P
1/16 (20180101); C07F 9/65586 (20130101); C07D
473/18 (20130101); C07D 421/04 (20130101); A61P
31/18 (20180101); A61P 31/12 (20180101); C07D
473/00 (20130101); C07F 9/65616 (20130101) |
Current International
Class: |
C07D
421/00 (20060101); C07D 421/04 (20060101); C07F
9/00 (20060101); C07F 9/6561 (20060101); C07D
473/18 (20060101); C07D 473/00 (20060101); C07F
9/6558 (20060101); A61K 031/505 (); C07D
239/02 () |
Field of
Search: |
;544/317 ;514/274 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 328 526 |
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Aug 1990 |
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EP |
|
0 337 713 B1 |
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Oct 1995 |
|
EP |
|
91/09124 |
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Jun 1991 |
|
WO |
|
WO 91/11186 |
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Aug 1991 |
|
WO |
|
WO 92/10497 |
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Jun 1992 |
|
WO |
|
WO 92/10496 |
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Jun 1992 |
|
WO |
|
92/15309 |
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Sep 1992 |
|
WO |
|
WO 92/14743 |
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Sep 1992 |
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WO |
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92/15308 |
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Sep 1992 |
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WO |
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92/18517 |
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Oct 1992 |
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WO |
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WO 94/04154 |
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Mar 1994 |
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WO |
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Other References
Du et. al., "Synthesis. Anti-Human Immunodeficiency Virus, and . .
. ", J. Med. Chem., Sep. 12, 1997, vol. 40(19), pp. 2991-2993.
.
Chang, et al., Deoxycytidine Deaminase-resistant Stereoisomer is
the Active Form of (-)-2',3'-thiacytidine in the Inhibition of
Hepatitis B Virus Replication, Journal of Biological Chemistry,
vol. 267(20), 13938-13942 (1992). .
Davisson, et al., Synthesis of Nucleotide 5'-Diphosphates from
5'-O-Tosyl Nucleoside, J. Org. Chem., 52(9), 1794-1801 (1987).
.
Du J et al, Synthesis, "Anti-Human Immunodeficiency Virus and
Anti-Hepatitis B Virus Activities of Novel Oxaselenolane
Nucleosides," J of Med. Chem., (40)19, 2991-2993 (1997). .
Furman, et al., "The Anti-Hepatitis B Virus Activities,
Cytotoxicities, and Anabolic Profiles of the (-) and (+)
Enantiomers of
cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathioloane-5-yl]-Cytosine"
Antimicrobial Agents and Chemotherapy,36(12) 2686-2692 (1992).
.
Ho, D.H.W., Distribution of Kinase and deaminase of 1
-D-rabinofuranosylcytosine in tissues of man and mouse. Cancer Res.
33, 2816-2820; (1973). .
Hoard, et al., Conversion of Mono- and Oligodeoxyribonucleotides to
5'-Triphosphates, J. Am. Chem. Soc., 87(8), 1785-1788 (1965). .
Hong, C.I., Nechaev, A., and West, C.R. (1979a) Synthesis and
antitumor activity of 1 -3-arabinofuranosylcytosine conjugates of
cortisol and cortisone. Biochem. Biophys. Rs. Commun. 88,
1223-1229(1979). .
Hong, C.I., Nechaev, A., Kirisits, A.J. Buchheit, D.J. and West,
C.R. (1980) Nucleoside conjugates. 6. Synthesis and comparison of
antitumor activity of 1-(-D-arabinofuranosyl) cytosine conjugates
of corticosteriods and selected lipophilic alcohols. J. Med. Chem.
28, 171-177. .
Ji, Y.H., Monophosphoric Acid Diesters of 7 -Hydroxycholesterol and
of Pyrimidine Nucleosides as Potential Antitumor Agents: Synthesis
and preliminary Evaluation of Antitumor Activity, J. Med. Chem.,
33,2264-2270, (1990). .
Hostetler, K.Y., Korba, B. Sridhar, C., Gardener, M., Antiviral
activity of phosphatidyl-dideoxycytidine in hepatitis B-infected
cells and enhanced hepatic uptake in mice. Antiviral Res. 24,
59-67; (1994). .
R. Jones and N. Bischofberger, Mini Review: Nucleotide prodrugs,
Antiviral Research, 27, 1-17 (1995). .
Kataoka, S., Uchida, R. and Yamaji, N. (1991) A convenient
synthesis of adenosine 3',5' cyclic phosphate (cAMP) benzyl and
methyl triesters. Heterocycles 32, 1351-1356. .
Hostetler, K.Y., Richman, D.D., Sridhar, C.N. Felgner, P.L.,
Felgner, J., Ricci, J., Gerdener, M.F. Selleseth, D.W. and Ellis,
M.N., Phosphatidylazidothymidine and phosphatidyl-ddC: Assessment
of uptake in mouse lymphoid tissues and antiviral activities in
human immunodeficiency virus-infected cells and in rauscher
leukemia virus-infected mice. Antimicrobial Agents Chemother. 38
(12), 2792-2797; (1994). .
Kinchington, D., Harvey, J.J., O'Connor, T.J., Jones, B.C.N.M.,
Devine, K.G., Taylor-Robinson, D., Jeffries, D.J. and McGuigan, C.
(1992) Comparison of antiviral effects of zidovudine
phosphoramidate and phosphorodiamidate derivatives against HIV and
ULV in vitro. Antiviral Chem. Chemother. 3, 107-112. .
Kodama, K., Morozumi, M., Saitoh, K.I., Kuninaka, H., Yoshino, H.
and Saneyoshi, M., Antitumor activity and pharmacology of
1--D-arabinofuranosylcytosine-5'-stearylphosphate; an orally active
derivative of 1--D-arabinofuranosylcytosine. .
Korba and Milman, A cell culture assay for compounds which inhibit
hepatitis B virus replication, Antiviral Res., 15:217 1991. .
Kumar, A., Goe, P.L., Jones, A.S. Walker, R.T. Balzarini, J. and De
Clercq, E. (1990) Synthesis and biological evaluation of some
cyclic phosphoramidate nucleoside derivatives. J. Med. Chem. 33,
2368-2375. .
LeBec, C., and Huynh-dinh, T., Synthesis of lipophilic phosphate
triester derivatives of 5-fluorouridine and arabinocytidine as
anticancer prodrugs, Tetrehedron Lett. 32, 6553-6556 (1991). .
Lichtenstein, J., Barner, H.D. and Cohen S.S., The metabolism of
exogenously supplied nucleotides by Escherichia coli., J. Biol.
Chem. 235, 457-465; (1960). .
McDougal, et al., Immunoassay for the Detection and Quanititation
of Infectious Human Retrovirus. Lymphadenopathy-Associated Virus
(LAV), J. Immun. Meth. 76, 171-183, (1985). .
McGuigan, C. Tollerfield, S.M. and Riley, P.A., Synthesis and
biological evaluation of some phosphate triester derivatives of the
anti-viral drug Ara. Nucleic Acids Res. 17, 6065-6075; (1989).
.
McGuigan, C., Pathirana, R.N., Mahmood, N., Devine, K.G. and Hay,
A.J., Aryl phosphate derivatives of AZT retain activity against
HIV1 in cell lines which are resistant to the action of AZT.
Antiviral Res. 17, 311-321 (1992). .
McGuigan, C., O'Connor, T.J., Nicholls, S.R. Nickson, C. and
Kinchington, D., Synthesis and anti-HIV activity of some novel
substituted dialkyl phosphate derivatives of AZT and ddCyd.,
Antiviral Chem. Chemother. 1, 355-360; (1990). .
McGuigan, C., Pathirana, R.N., Choi, S.M., Kinchington, D. and
O'Connor, T.J., Phosphoramidate derivatives of AZT as inhibitors of
HIV; studies on the caroxyl terminus. Antiviral Chem. Chemother. 4,
97-101; (1993). .
McGuigan, C., Pathirana, R.N., Balzarini, J. and De Clercq, E.
Intracellular delivery of bioactive AZT nucleotides by aryl
phosphate derivatives of AZT. J. Med. Chem. 36, 1048-1052 (1993).
.
Meyer, R.B., Jr., Shuman, D.A. and Robins, R.K., Synthesis of
purine nucleoside 3',5'-cyclic phosphoramidates. Tetrahedron Lett.,
269-272; (1973). .
Namane, A. Goyette, C., Fillion, M.P., Fillion, G. and Huynh-Dinh,
T. (1992) Improved brain delivery of AZT using a glycosyl
phosphotriester prodrug. J. Med. Chem. 35, 3939-3044. .
Nargeot, J. Nerbonne, J.M. Engels, J. and Leser, H.A., Time course
of the increase in the myocardial slow inward current after a
photochemically generated concentration jump of intracelluar aAMP,
Natl. .
Norbeck, Tetrahedron Letters 30 (46), 6246 (1989). .
Ohno, R., Tatsumi, N., Hirano, M., Imai, K. Mizoguchi, H.,
Nakamura, T., Kosaka, M., Takatuski, K., Yamaya, T., Toyama, K.,
Yoshida, T., Masaoka, T., Hashimoto, S., Ohshima, T., Kimura, I.,
Yamada, K. and Kimura, J., Treatment of myelodyspastic syndromes
with orally administred
1--D-rabinofuranosylcytosine-5'-stearylphosphate. Oncology 48,
451-455 (1991). .
Palomino, E., Kessle, D. and Horwitz, J.P., A dihydropyridine
carrier system for sustained delivery of 2',3'dideoxynucleosides to
the brain., J. Med. Chem. 32, 622-625; (1989). .
Piantadosi, C., Marasco, C.J., Jr., Morris-Natschke, S.L., Meyer,
K.L., Gumus, F., Surles, J.R., Ishaq, K.S., Kucera, L.S. Iyer, N.,
Wallen, C.A., Piantodosi, S. and Modest, E.J. (1991) Synthesis and
evaluation of novel ether lipid nucleoside conjugates for
anti-HIV-1 activity. J. Med. Chem. 34, 1408-1414. .
Prisbe, E.J., Martin, J.C., McGee, D.P.C., Barker, M.F., Smee, D.F.
Duke, A.E., Matthews, T.R. and Verheyden, J.P.J. (1986) Synthesis
and antiherpes virus activity of phosphate and phosphonate
derivatives of 9-[(1,3-dihydroxy-2-propoxy)methyl] guanine. J. Med.
Chem. 29, 671-675. .
Philpott, M.S., Ebner, J.P., Hoover, E.A., Evaluation of
9-(2-phosphonylmethoxyethyl) adenine therapy for feline
immunodeficiency virus using a quantitative polymerase chain
reaction, Vet. Immunol. Immunopathol. 35:155-166, (1992). .
Puech, F., Gosselin, G., Lefebvre, I., Pompon, A., Aubertin, A.M.
Dirn, A. and Imbach, J.L. (1993) Intracellular delivery of
nucleoside monophosphate through a reductase-mediated activation
process. Antiviral Res. 22, 155-174. .
Rosowsky, A., Kim, S.H., Ross and J. Wick, M.M., Lipophilic
5'-(alkylphosphate) esters of 1--D-arabinofuranosylcytosine and its
N4-acyl and 2.2'-anhydro-3'0-acyl derivatives as potential
prodrugs. J. Med. Chem. 25, 171-178; (1982). .
Ryu, E.K., Ross, R.J., Matsushita, T., MacCoss, M., Hong, C.I. and
West, C.R, Phospholipid-nucleoside conjugates 3. Synthesis and
preliminary biological evaluation of 1--D-arabinofuranosylcytosine
5'diphosphate[=], 2-diacylglycerols. J. Med. Chem. 25, 1322-1329;
(1982). .
Saffhill, R. and Hume, W.J., The degradation of 5-iododeoxyurindine
and 5-bromoeoxyuridine by serum from different sources and its
consequences for the use of these compounds for incorporation into
DNA. Chem. Biol. Interact. 57, 347-355; (1986). .
Sastry, J.K., Nehete, P.N., Khan, S., Nowak, B.J., Plunkett, W.,
Arlinghaus, R.B. and Farquhar, D., Membrane-permeable
dideoxyuridine 5'-monophosphate analogue inhibits human
immunodeficiency virus infection. Mol. Pharmacol. 41, 441-445;
(1992). .
Schinazi, et al., Mutations in retroviral genes associated with
drug resistance, International Antiviral News, vol. 1(6),
International Medical Press(1996). .
Schinazi, et al., Antimicrob. Agents Chemother. 34:1061-1067
(1990). .
Schinazi, et al., Antimicrob. Agents Chemother. 32, 1784-1787
(1988). .
Schinazi, et al., Effect of Combinations of Acylovir with
Vidarabine or its Monophosphate on Herpes Simplex Viruses in Cell
Culture and in Mice, Antimicrobial Agents and Chemotherapy, 22 (3),
499, (1982). .
Schinazi, et al., Selective Inhibition of Human Immunodeficiency
Viruses by Racemates and Enantiomers of
cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-Oxathiolane-5-yl] Cytosine,
Antimicrobial Agents and Chemotherapy,36 (11), 2423-2431 (1992).
.
Shuto, S., Ueda, S., Imamura, S., Fukukuawa, K. Matsuda, A. and
Ueda, T., A facile one-step synthesis of 5'-phosphatidylnucleosides
by an enzymatic two-phase reaction. Tetrahedron Lett. 28, 199-202;
(1987). .
Wang, S., Montelaro, R., Schinazi, R.R., Jagerski, B. and Mellors,
J.W.: Activity of nucleoside and non-nucleoside reverse
transcriptase inhibitors (NNRTI) against equine infectious anemia
virus (EIAV). First National Conference on Human Retroviruses and
Related Infections, Washington, DC, Dec. 12-16, 1993..
|
Primary Examiner: Raymond; Richard L.
Assistant Examiner: Truong; Tamthom N.
Attorney, Agent or Firm: Knowles; Sherry M. Haley;
Jacqueline King & Spalding
Government Interests
The U.S. government has rights in this invention resulting from
U.S. Public Health Service Research grants from the National
Institute of Allergy and Infectious Diseases and the Department of
Veterans Affairs which partially funded the research leading to
this invention.
Parent Case Text
This application claims priority to U.S. provisional patent
application
Ser. No. 60/041,265, filed on Mar. 19, 1997.
Claims
We claim:
1. A 1,3-oxaselenolane nucleoside of the formula: ##STR2## wherein
B is a pyrimidine base, and R is hydrogen, acyl, a mono-, di- or
triphosphate ester, a stabilized phosphate, or an ether lipid, or a
pharmaceutically acceptable salt thereof, and wherein the
nucleoside exhibits an EC.sub.50 of less than 10 micromolar in
HIV-infected PBM cells.
2. The 1,3-oxaselenolane nucleoside of claim 1, wherein B is a
pyrimidine base.
3. The 1,3-oxaselenolane nucleoside of claim 2, wherein R is
hydrogen.
4. The 1,3-oxaselenolane nucleoside of claim 2, wherein R is
acyl.
5. The 1,3-oxaselenolane nucleoside of claim 2, wherein R is
monophosphate.
6. The 1,3-oxaselenolane nucleoside of claim 2, wherein R is
diphosphate.
7. The 1,3-oxaselenolane nucleoside of claim 2, wherein R is
triphosphate.
8. The 1,3-oxaselenolane nucleoside of claim 2, wherein R is a
stabilized phosphate.
9. The 1,3-oxaselenolane nucleoside of claim 2, wherein R is a
lipid ether.
10. The 1,3-oxaselenolane nucleoside of claim 1, wherein B is
cytosine.
11. The 1,3-oxaselenolane nucleoside of claim 1, wherein B is
5-fluorocytosine.
12. The 1,3-oxaselenolane nucleoside of claim 1, which is
2-hydroxymethyl-4-(N-5'-cytosin-1'-yl)-1,3-oxaselenolane, or a
pharmaceutically acceptable salt thereof.
13. The 1,3-oxaselenolane nucleoside of claim 1, which is
2-hydroxymethyl-4-(N-5'-fluorocytosin-l'-yl)-1,3-oxaselenolane, or
a pharmaceutically acceptable salt thereof.
14. The 1,3-oxaselenolane nucleoside of claim 1, which is
(-)-.beta.-L-2-hydroxymethyl-4-(N-5'-cytosin-l'-yl)-1,3-oxaselenolane
as an isolated enantiomer, or a pharmaceutically acceptable salt
thereof.
15. The 1,3-oxaselenolane nucleoside of claim 1, which is
(-)-.beta.-L-2-hydroxymethyl-4-(N-5'-fluorocytosin-1'-yl)-1,3-oxaselenolan
e as an isolated enantiomer, or a pharmaceutically acceptable salt
thereof.
16. A pharmaceutical composition for the treatment of HIV or HBV
infection in humans and other host animals, comprising an effective
amount of a 1,3-oxaselenolane nucleoside of claim 1 together with a
pharmaceutically acceptable carrier.
17. A pharmaceutical composition for the treatment of HIV or HBV
infection in humans and other host animals, comprising an effective
amount of a 1,3-oxaselenolane nucleoside of claim 2 together with a
pharmaceutically acceptable carrier.
18. A pharmaceutical composition for the treatment of HIV or HBV
infection in humans and other host animals, comprising an effective
amount of a 1,3-oxaselenolane nucleoside of one of claims 4-12 or
14-17 together with a pharmaceutically acceptable carrier.
19. A method for treating HIV in humans comprising administering an
HIV-effective amount of a 1,3-oxaselenolane nucleoside of the
formula ##STR3## wherein B is a pyrimidine base, and R is hydrogen,
acyl, or a mono-, di-, or triphosphate ester, a stabilized
phosphate, or an ether lipid, or a pharmaceutically acceptable salt
thereof, and wherein the nucleosides exhibits an EC.sub.50 of less
than 10 micromolar in HIV-infected PBM cells.
20. The method for the treatment of HIV of claim 19, wherein B is a
pyrimidine base.
21. The method for the treatment of HIV of claim 19, wherein R is
hydrogen.
22. The method for the treatment of HIV as claimed in claim 19,
wherein R is acyl.
23. The method for the treatment of HIV as claimed in claim 19,
wherein R is monophosphate.
24. The method for the treatment of HIV as claimed in claim 19,
wherein R is diphosphate.
25. The method for the treatment of HIV as claimed in claim 19,
wherein R is triphosphate.
26. A method for the treatment of HIV as claimed in claim 19,
wherein R is hydrogen.
27. A method for the treatment of HIV as claimed in claim 19,
wherein R is acyl.
28. A method for the treatment of HIV as claimed in claim 19,
wherein R is monophosphate.
29. A method for the treatment of HIV as claimed in claim 19,
wherein R is ditriphosphate.
30. A method for the treatment of HIV as claimed in claim 20,
wherein the 1,3-oxaselenolane nucleoside is
2-hydroxymethyl-4-(N-5'-cytosin-1'-yl)-1,3-oxaselenolane, or a
pharmaceutically acceptable salt thereof.
31. A method for the treatment of HIV as claimed in claim 20,
wherein the 1,3-oxaselenolane nucleoside is
2-hydroxymethyl-4-(N-5'-fluorocytosin-1'-yl)-1,3-oxaselenolane, or
a pharmaceutically acceptable salt thereof.
32. A method for the treatment of HIV as claimed in claim 20,
wherein the 1,3-oxaselenolane nucleoside is
(-)-.beta.-L-2-hydroxymethyl-4-(N-5'-cytosin-1'-yl)-1,3-oxaselenolane
as an isolated enantiomer, or a pharmaceutically acceptable salt
thereof.
33. A method for the treatment of HIV as claimed in claim 20,
wherein the 1,3-oxaselenolane nucleoside is
(-)-.beta.-L-2-hydroxymethyl-4-(N-5'-fluorocytosin-1'-yl)-1,3-oxaselenolan
e as an isolated enantiomer, or a pharmaceutically acceptable salt
thereof.
34. A method for treating hepatitis B in humans and other host
animals comprising administering a hepatitis B-effective amount of
a 1,3-oxaselenolane nucleoside of the formula ##STR4## wherein B is
a pyrimidine base, and R is hydrogen, acyl, or a mono-, di-, or
triphosphate ester, a stabilized phosphate, or an ether lipid, or a
pharmaceutically acceptable salt thereof, in racemic form or as an
isolated enantiomer, and wherein the nucleoside exhibits an
EC.sub.50 of less than 10 micromolar in HBV-transfected 2.2.15
cells.
35. A method for the treatment of HBV as claimed in claim 34,
wherein B is a pyrimidine base.
36. A method for the treatment of HBV as claimed in claim 34,
wherein R is hydrogen.
37. A method for the treatment of HBV as claimed in claim 34,
wherein R is acyl.
38. A method for the treatment of HBV as claimed in claim 34,
wherein R is monophosphate.
39. A method for the treatment of HBV as claimed in claim 34,
wherein R is ditriphosphate.
40. A method for the treatment of HBV as claimed in claim 34,
wherein R is triphosphate.
41. A method for the treatment of HBV as claimed in claim 34,
wherein R is hydrogen.
42. A method for the treatment of HBV as claimed in claim 34,
wherein R is acyl.
43. A method for the treatment of HBV as claimed in claim 34,
wherein R is monophosphate.
44. A method for the treatment of HBV as claimed in claim 34,
wherein R is diphosphate.
45. A method for the treatment of HBV as claimed in claim 34,
wherein R is triphosphate.
46. A method for the treatment of HBV as claimed in claim 34,
wherein the 1,3-oxaselenolane nucleoside is
2-hydroxymethyl-4-(N-5'-cytosin-1'-yl)-1,3-oxaselenolane, or a
pharmaceutically acceptable salt thereof.
47. A method for the treatment of HBV as claimed in claim 34,
wherein the 1,3-oxaselenolane nucleoside is
2-hydroxymethyl-4-(N-5'-fluorocytosin-1'-yl)-1,3-oxaselenolane, or
a pharmaceutically acceptable salt thereof.
48. A method for the treatment of HBV as claimed in claim 34,
wherein the 1,3-oxaselenolane nucleoside is
(-)-.beta.-L-2-hydroxymethyl-4-(N-5'-cytosin-1'-yl)-1,3-oxaselenolane
as an isolated enantiomer, or a pharmaceutically acceptable salt
thereof.
49. A method for the treatment of HBV as claimed in claim 34,
wherein the 1,3-oxaselenolane nucleoside is
(-)-.beta.-L-2-hydroxymethyl-4-(N-5'-fluorocytosin-1'-yl)-1,3-oxaselenolan
e as an isolated enantiomer, or a pharmaceutically acceptable salt
thereof.
Description
BACKGROUND OF THE INVENTION
This invention is in the area of synthetic nucleosides, and is
specifically directed to 1,3-oxaselenolane nucleosides and their
pharmaceutical uses, compositions, and method of preparation.
In 1981, acquired immune deficiency syndrome (AIDS) was identified
as a disease that severely compromises the human immune system, and
that almost without exception led to death. In 1983, the
etiological cause of AIDS was determined to be the human
immunodeficiency virus (HIV).
In 1985, it was reported that the synthetic nucleoside
3'-azido-3'-deoxythymidine (AZT) inhibits the replication of human
immunodeficiency virus. Since then, a number of other synthetic
nucleosides, including 2',3'-dideoxyinosine (DDI),
2',3'-dideoxycytidine (DDC), 2',3'-dideoxy-2',3'-didehydrothymidine
(D4T), and
(1S,4R)-4-[2-amino-6-cyclopropyl-amino)-9H-purin-9-yl]-2-cyclopentene-1-me
thanol succinate ("159U89"), have been proven to be effective
against HIV. In general, after cellular phosphorylation to the
5'-triphosphate by cellular kinases, these synthetic nucleosides
are incorporated into a growing strand of viral DNA, causing chain
termination due to the absence of the 3'-hydroxyl group. They can
also or alternatively inhibit the viral enzyme reverse
transcriptase or DNA polymerase.
The success of various synthetic nucleosides in inhibiting the
replication of HIV in vivo or in vitro has led a number of
researchers to design and test nucleosides that substitute a
heteroatom for the carbon atom at the 3'-position of the
nucleoside. Norbeck, et al., disclosed that
(.+-.)-1-[(2.beta.,4.beta.)-2-(hycroxymethyl)-4-dioxolanyl]thymine
(referred to as (.+-.)-dioxolane-T) exhibits a modest activity
against HIV (EC.sub.50 of 20 .mu.M in ATH8 cells), and is not toxic
to uninfected control cells at a concentration of 200 .mu.M.
Tetrahedron Letters 30 (46), 6246, (1989). European Patent
Application Publication No. 0 337 713 and U.S. Pat. No. 5,041,449,
assigned to BioChem Pharma, Inc., disclose racemic
2-substituted-4-substituted-1,3-dioxolanes that exhibit antiviral
activity. Published PCT applications PCT/US91/09124 and
PCT/US93/08044 disclose purified .beta.-D-1,3-dioxolanyl
nucleosides for the treatment of HIV infection. PCT discloses the
use of purified .beta.-D-1,3-dioxolanyl nucleosides for the
treatment of HBV infection.
PCT/US95/11464 discloses that
(-)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine is useful
in the treatment of tumors and other abnormal cell
proliferation.
U.S. Pat. No. 5,047,407 and European Patent Application Publication
No. 0 382 526, both to BioChem Pharma, Inc., disclose that a number
of racemic 2-substituted-5-substituted-1,3-oxathiolane nucleosides
have antiviral activity, and specifically report that the racemic
mixture of 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane
(referred to below as BCH-189) has approximately the same activity
against HIV as AZT, with less toxicity. U. S. Pat. No. 5,539,116 to
Liotta, et al., directed to the (-)-enantiomer of BCH-189, known as
3TC, is now sold commercially for the treatment of HIV in humans in
the United States.
It has also been disclosed that
cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane
("FTC") has potent HIV activity. Schinazi, et al., "Selective
Inhibition of Human Immunodeficiency viruses by Racemates and
Enantiomers of
cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-Oxathiolane-5-yl]Cytosine"
Antimicrobial Agents and Chemotherapy, November 1992, page
2423-2431. See also U.S. Pat. No. 5,210,085; U.S. Pat. No.
5,204,466, WO 91/11186, and WO 92/14743.
Another virus that causes a serious human health problem is the
hepatitis B virus (referred to below as "HBV"). HBV is second only
to tobacco as a cause of human cancer. The mechanism by which HBV
induces cancer is unknown. It is postulated that it may directly
trigger tumor development, or indirectly trigger tumor development
through chronic inflammation, cirrhosis, and cell regeneration
associated with the infection.
After a two to six month incubation period in which the host is
unaware of the infection, HBV infection can lead to acute hepatitis
and liver damage, that causes abdominal pain, jaundice, and
elevated blood levels of certain enzymes. HBV can cause fulminant
hepatitis, a rapidly progressive, often fatal form of the disease
in which massive sections of the liver are destroyed.
Patients typically recover from acute hepatitis. In some patients,
however, high levels of viral antigen persist in the blood for an
extended, or indefinite, period, causing a chronic infection.
Chronic infections can lead to chronic persistent hepatitis.
Patients infected with chronic persistent HBV are most common in
developing countries. By mid-1991, there were approximately 225
million chronic carriers of HBV in Asia alone, and worldwide,
almost 300 million carriers. Chronic persistent hepatitis can cause
fatigue, cirrhosis of the liver, and hepatocellular carcinoma, a
primary liver cancer.
In western industrialized coutrines, high risk groups for HBV
infection include those in contact with HBV carriers or their blood
samples. The epidemiology of HBV is very similar to that of
acquired immune deficiency syndrome, which accounts for why HBV
infection is common among patients with AIDS or AIDS related
complex. However, HBV is more contagious than HIV.
Both FTC and 3TC exhibit activity against HBV. See Furman, et al.,
"The Anti-Hepatitis B Virus Activities, Cytotoxicities, and
Anabolic Profiles of the (-) and (+) Enantiomers of
cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]-Cytosine"
Antimicrobial Agents and Chemotherapy, l December 1992, page
2686-2692; and Cheng, et al., Journal of Biological Chemistry,
Volume 267(20), 13938-13942 (1992).
A human serum-derived vaccine has been developed to immunize
patients against HBV. However, more recently, vaccines have also
been produced through genetic engineering and are currently used
widely. Unfortunately, vaccines cannot help those already infected
with HBV. Daily treatment with a-interferon, a genetically
engineered protein, has also shown promise, but this therapy is
only successful in about one third of treated patients. Further,
interferon cannot be given orally.
Since 1,3-dioxolane and 1,3-oxathiolane nucleosides have exhibited
promising antiviral and anticancer activities, it was of interest
to synthesize an isosteric class of compounds, 1,3-oxaselenolane
nucleosides in search of biologically interesting nucleosides.
Despite their structural similarity to the 3'-heteroatom
substituted nucleosides, the synthesis of 1,3-oxaselenolane
nucleosides has been elusive as the construction of the
oxaselenolane ring is difficult. For this reason, it appears that
1,3-oxaselenolane nucleosides have never been reported.
In light of the fact that acquired immune deficiency syndrome,
AIDS-related complex, and hepatitis B virus have reached epidemic
levels worldwide, and have tragic effects on the infected patient,
there remains a strong need to provide new effective pharmaceutical
agents to treat these diseases.
Therefore, it is an object of the present invention to provide a
method and composition for the treatment of human patients infected
with HIV.
It is another object of the present invention to provide a method
and composition for the treatment of human patients or other host
animals infected with HBV.
It is a further object of the invention to provide a method for the
synthesis of 1,3-oxaselenolanyl nucleosides.
It is a still further object of the invention to provide
1,3-oxaselenolanyl nucleosides and pharmaceutical compositions that
include 1,3-oxaselenolanyl nucleosides.
SUMMARY OF THE INVENTION
A method and composition for the treatment of HIV or HBV infection
in humans and other host animals is disclosed that includes the
administration of an effective amount of a 1,3-oxaselenolane
nucleoside or a pharmaceutically acceptable salt thereof,
optionally in a pharmaceutically acceptable carrier.
In one embodiment, the 1,3-oxaselenolane nucleoside has the
formula: ##STR1## wherein B is a purine or pyrimidine base, and R
is hydrogen, acyl or a phosphate ester, including monophosphate,
diphosphate, or triphosphate. In another embodiment, the
1,3-oxaselenolanyl nucleoside is provided as a lipophilic or
hydrophilic prodrug as discussed in more detail below. In another
alternative embodiment, the selenium atom is oxidized in the
molecule. Preferred 1,3-oxaselenolanyl nucleosides are those that
exhibit an activity against HIV or HBV at a concentration of less
than approximately 10 micromolar, and most preferably approximately
5 micromolar or less in an in vitro assay such as those described
in detail in this application. For treatment of HIV and HBV, it is
also preferred that the 1,3-oxaselenolanyl nucleoside exhibit an
IC.sub.50 toxicity in an in vitro assay such as those described
herein of greater than 50 micromolar, and more preferably,
approximately 100 micromolar or greater.
The 1,3-oxaselenolane nucleoside is preferably either a
.beta.-L-nucleoside or a .beta.-D-nucleoside, as an isolated
enantiomer. In one embodiment, the nucleoside is a .beta.-L- or
.beta.-D-nucleoside in substantially pure form, i.e., substantially
in the absence of the corresponding .beta.-D- or
.beta.-L-nucleoside.
Preferred compounds are
2-hydroxymethyl-4-(N-5'-cytosin-1'-yl)-1,3-oxaselenolane and
2-hydroxymethyl-4-(N-5'-fluorocytosin-1'-yl)-1,3-oxaselenolane. It
has been discovered that the isolated (-)-.beta.-L-enantiomer of
these nucleosides are more potent than their .beta.-D counterparts.
The (+)-enantiomers of these compounds, however, are not toxic to
CEM cells.
In another embodiment, the active compound or its derivative or
salt can be administered in combination or alternation with another
antiviral agent, such as an other anti-HIV agent or anti-HBV agent,
as described in more detail in Section IV. In general, during
alternation therapy, an effective dosage of each agent is
administered serially, whereas in combination therapy, an effective
dosage of two or more agents are administered together. The dosages
will depend on absorption, inactivation, and excretion rates of the
drug as well as other factors known to those of skill in the art.
It is to be noted that dosage values will also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens and schedules should be adjusted over time according to
the individual need and the professional judgment of the person
administering or supervising the administration of the
compositions.
The compounds can also be used to treat equine infectious anemia
virus (EIAV), feline immunodeficiency virus, and simian
immunodeficiency virus. (Wang, S., Montelaro, R., Schinazi, R. R.,
Jagerski, B. and Mellors, J. W.: Activity of nucleoside and
non-nucleoside reverse transcriptase inhibitors (NNRTI) against
equine infectious anemia virus (EIAV). First National Conference on
Human Retroviruses and Related Infections, Washington, DC, Dec.
12-16, 1993; Sellon D. C., Equine Infectious Anemia, Vet. Clin.
North Am. Equine Pract. United States, 9: 321-336, 1993; Philpott,
M. S., Ebner, J. P., Hoover, E. A., Evaluation of
9-(2-phosphonylmethoxyethyl) adenine therapy for feline
immunodeficiency virus using a quantitative polymerase chain
reaction, Vet. Immunol. Immunopathol. 35:155166, 1992.)
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is an illustration of one process for the preparation of a
1,3-oxaselenolanyl nucleoside according to the present invention,
as described in Example 1.
FIG. 2 is an illustration of one process for the preparation of
.beta.-D and .beta.-L 1,3-oxaselenolanyl nucleosides according to
the present invention, as described in Example 3.
FIG. 3 is the x-ray crystal structure of
[2-(1'R,2'S,5'R)-menthyl-(5-one-1,3-oxaselenolane)]-L-carboxylate.
FIG. 4 is an illustration of the structures of the enantiomers of
(+)-.beta.-Se-ddC, (-)-.beta.-Se-ddC, (+)-.beta.-Se-FddC and
(-)-.beta.-Se-FddC.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "isolated enantiomer" refers to a
nucleoside composition that includes at least approximately 95% to
100%, or more preferably, over 97% of a single enantiomer of that
nucleoside.
The term "substantially pure form" refers to a nucleoside
composition of one enantiomer that includes no more than about 5%
w/w of the other enantiomer, more preferably no more than about 2%,
and most preferably less than about 1% w/w is present.
The term purine or pyrimidine base, includes, but is not limited
to, N.sup.6 -alylpurines, N.sup.6 -acylpurines, N.sup.6
-benzylpurine, N.sup.6 -halopurine, N.sup.6 -vinylpurine, N.sup.6
-acetylenic purine, N.sup.6 -acyl purine, N.sup.6 -hydroxyalkyl
purine, N.sup.6 -thioalkyl purine, N.sup.2 -alkylpurines, N.sup.4
-alkylpyrimidines, N.sup.4 -acylpyrimidines, N.sup.4 -benzylpurine,
N.sup.4 -halopyrimidines, N.sup.4 -vinylpyrimidines, N.sup.4
-acetylenic pyrimidines, N.sup.4 -acyl pyrimidines, N.sup.4
-hydroxyalkyl pyrimidines, N.sup.6 -thioalkyl pyrimidines, thymine,
cytosine, 6-azapyrimidine, including 6-azacytosine, 2-and/or
4-mercaptopyrimidine, uracil, C.sup.5 -alkylpyrimidines, C.sup.5
-benzylpyrimidines, C.sup.5 -halopyrimidines, C.sup.5
-vinylpyrimidine, C.sup.5 -acetylenic pyrimidine, C.sup.5 -acyl
pyrimidine, C.sup.5 -hydroxyalkyl purine, C.sup.5 -amidopyrimidine,
C.sup.5 -cyanopyrimidine, C'-nitropyrimidine, C.sup.5
-aminopyrimdine, N.sup.2 -alkylpurines, N.sup.2
-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,
trazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and
pyrazolopyrimidinyl. Functional oxygen and nitrogen groups on the
base can be protected as necessary or desired. Suitable protecting
groups are well known to those skilled in the art, and included
trimethylsilyl, dimethylhexylsilyl, t-butyldimenthylsilyl, and
t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as
acetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.
Preferred bases include cytosine, 5-fluorocytosine, uracil,
thymine, adenine, guanine, xanthine, 2,6-diaminopurine,
6-aminopurine, and 6-chloropurine.
The term alkyl, as used herein, unless otherwise specified, refers
to a saturated straight, branched, or cyclic, primary, secondary,
or tertiary hydrocarbon, typically of C.sub.1 to C.sub.18, and
specifically includes methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl,
hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl,
2,2-dimethylbutyl, and 2,3-dimethylbutyl. The alkyl group can be
optionally substituted with one or more moieties selected from the
group consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy,
aryloxy, nitro, cyano, sulfonic acid, sulfate, phophonic acid,
phosphate, or phosphonate, either unprotected, or protected as
necessary, as known to those skilled in the art, for example, as
taught in Greene, et al., "Protective Groups in Organic Synthesis,"
John wiley and Sons, Second Edition, 1991, hereby incorporated by
reference.
The term lower alkyl, as used herein, and unless otherwise
specified, refers to a C.sub.1 to C.sub.4 saturated straight or
branched alkyl group.
The term "protected" as used herein and unless otherwise defined
refers to a group that is added to an oxygen, nitrogen, or
phosphorus atom to prevent its further reaction or for other
purposes. A wide variety of oxygen and nitrogen protecting groups
are known to those skilled in the art of organic synthesis.
The term aryl, as used herein, and unless otherwise specified,
refers to phenyl, biphenyl, or naphthyl, and preferably phenyl. The
aryl group can be optionally substituted with one or more moieties
selected from the group consisting of hydroxyl, halo, alkyl,
alkenyl, alkynyl, alkaryl aralkyl, amino, alkylamino, alkoxy,
aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,
phosphate, or phosphonate, either unprotected,
or protected as necessary, as known to those skilled in the art,
for example, as taught in Greene, et al., "Protective Groups in
Organic Synthesis," John Wiley and Sons, Second Edition, 1991.
The term alkaryl or alkylaryl refers to an alkyl group with an aryl
substituent.
The term aralkyl or arylalkyl refers to an aryl group with an alkyl
substituent.
The term halo, as used herein, includes chloro, bromo, iodo, and
fluoro.
The term acyl refers to moiety of the formula --C(O)R', wherein R'
is alkyl, aryl, alkaryl, aralkyl, heteroaromatic, alkoxyalkyl
including methoxymethyl; arylalkyl including benzyl; aryloxyalkyl
such as phenoxymethyl; aryl including phenyl optionally substituted
with halogen, C.sub.1 to C.sub.4 alkyl or C.sub.1 to C.sub.4
alkoxy, or the residue of an amino acid.
As used herein, a leaving group means a functional group that is
cleaved from the molecule to which it is attached under appropriate
conditions.
The term amino acid includes naturally occurring and synthetic
amino acids, and includes but is not limited to, alanyl, valinyl,
leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl,
methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,
asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl,
and histidinyl.
The term heteroaryl or heteroaromatic, as used herein, refers to an
aromatic moiety that includes at least one sulfur, oxygen, or
nitrogen in the aromatic ring. Nonlimiting examples are furyl,
pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl,
pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl,
benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl,
benzimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl,
isothiazolyl, 1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl,
quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl, phthalazinyl,
quinoxalinyl, xanthinyl, hypoxantinyl, and pteridinyl. Functional
oxygen and nitrogen groups on the heterocyclic base can be
protected as necessary or desired. Suitable protecting groups are
well known to those skilled in the art, and include trimethylsilyl,
dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl,
trityl or substituted trityl, alkyl groups, acycl groups such as
acetyl and propionyl, methanesulfonyl, and p-toluenelsulfonyl.
The term lipophilic prodrug refers to a 1,3-oxaselenolanyl
nucleoside that contains a covalent substituent that is cleavable
at the 5'-hydroxyl position that renders the nucleoside more
lipophilic than the parent nucleoside with a 5'-hydroxyl group.
The term hydrophilic prodrug refers to a 1,3-oxaselenolanyl
nucleoside that contains a covalent substitutent at the 5'-hydroxyl
position that renders the nucleoside more hydrophilic than the
parent nucleoside with a 5'-hydroxyl group.
The invention as disclosed herein is a method and composition for
the treatment of HIV or HBV infection, and other viruses infections
replicating in like manner, in humans or other host animals, that
includes administering an effective amount of a 1,3-oxaselenolanyl
nucleoside, a pharmaceutically acceptable derivative thereof,
including a 1,3-oxaselenolanyl nucleoside with a 5' leaving group,
including an acylated or phosphorylated derivative or a
pharmaceutically acceptable salt thereof, optionally in a
pharmaceutically acceptable carrier. The compounds of this
invention either possess antiviral activity, such as anti-HIV-1,
anti-HIV-2, anti-HBV, or anti-simian immunodeficiency virus
(anti-SIV) activity themselves or are metabolized to a compound
that exhibits antiviral activity.
The disclosed compounds or their pharmaceutically acceptable
derivatives or salts or pharmaceutically acceptable formulations
containing these compounds are useful in the prevention and
treatment of HIV infections and other related conditions such as
AIDS-related complex (ARC), persistent generalized lymphadenopathy
(PGL), AIDS-related neurological conditions, anti-HIV antibody
positive and HIV-positive conditions, Kaposi's sarcoma,
thrombocytopenia purpurea and opportunistic infections. In
addition, these compounds or formulations can be used
prophylactically to prevent or retard the progression of clinical
illness in individuals who are anti-HIV antibody or HIV-antigen
positive or who have been exposed to HIV.
The compound or its pharmaceutically acceptable derivatives or
salt, or pharmaceutically acceptable formulations containing the
compound or its derivatives or salt, are also useful in the
prevention and treatment of HBV infections and other related
conditions such as anti-HBV antibody positive and HBV-positive
conditions, chronic liver inflammation caused by HBV, cirrhosis,
acute hepatitis, fulminant hepatitis, chronic persistent hepatitis,
and fatigue. These compounds or formulations can also be used
prophylactically to prevent or retard the progression of clinical
illness in individuals who are anti-HBV antibody or HBV antigen
positive or who have been exposed to HBV.
The compound can be converted into a pharmaceutically acceptable
ester by reaction with an appropriate esterifying agents, for
example, an acid halide or anhydride. The compound or its
pharmaceutically acceptable derivative can be converted into a
pharmaceutically acceptable salt thereof in a conventional manner,
for example, by treatment with an appropriate base. The ester or
salt of the compound can be converted into the parent compound, for
example, by hydrolysis.
In summary, the present invention, includes the following
features:
(a) 1,3-oxaselenolane nucleosides as outlined above, and
pharmaceutically acceptable derivatives and salts thereof;
(b) 1,3-oxaselenolane nucleosides, and pharmaceutically acceptable
derivatives and salts thereof for use in medical therapy, for
example for the treatment or prophylaxis of an HIV or HBV
infection;
(c) use of 1,3-oxaselenolane nucleosides and pharmaceutically
acceptable derivatives and salts thereof in the manufacture of a
medicament for treatment of an HIV or HBV infection;
(d) pharmaceutical formulations comprising 1,3-oxaselenolane
nucleosides or a pharmaceutically acceptable derivative or salt
thereof together with a pharmaceutically acceptable carrier or
diluent;
(e) processes for the preparation of 1,3-oxaselenolane nucleosides;
and
(f) use of 1,3-oxaselenolanyl nucleosides in the treatment of viral
infections by administration in combination or alternation with
another antiviral agent.
I. Active Compound, and Physiological Acceptable Derivatives and
Salts Thereof
The active compounds disclosed herein are 1,3-oxaselenolane
nucleosides, in the racemic form or as isolated enantiomers.
The active compound can be administered as any derivative that upon
administration to the recipient, is capable of providing directly
or indirectly, the parent compound, or that exhibits activity
itself. Nonlimiting examples are the pharmaceutically acceptable
salts (alternatively referred to as "physiologically acceptable
salts"), and the 5' and N.sup.4 pyrimidine or N.sup.6 -purine
acylated or alkylated derivatives of the active compound
(alternatively referred to as "physiologically active
derivatives"). In one embodiment, the acyl group is a carboxylic
acid ester in which the non-carbonyl moiety of the ester group is
selected from straight, branched, or cyclic alkyl or lower alkyl,
alkoxyalkyl including methoxymethyl, aralkyl including benzyl,
aryloxyalkyl such as phenoxymethyl, aryl including phenyl
optionally substituted with halogen, C.sub.1 to C.sub.4 alkyl or
C.sub.1 to C.sub.4 alkoxy, sulfonate esters such as alkyl or
aralkyl sulphonyl including methanesulfonyl, phosphate, including
but not limited to mono, di or triphosphate ester, trityl or
monomethoxytrityl, substituted benzyl, trialkylsilyl (e.g.,
dimethyl-5-butylsilyl) or diphenylmethylsilyl. Aryl groups in the
esters optionally comprise a phenyl group.
Modifications of the active compound, and especially at the N.sup.4
pyrimidinyl or N.sup.6 purine and 5'--O positions, can affect the
bioavailability and rate of metabolism of the active species, thus
providing control over the delivery of the active species. Further,
the modifications can affect that antiviral activity of the
compound, in some cases increasing the activity over the parent
compound. This can easily be assessed by preparing the derivative
and testing its antiviral activity according to the methods
described herein, or other methods known to those skilled in the
art.
Nucleotide Prodrugs
Any of the nucleotides described herein can be administered as a
nucleotide prodrug to increase the activity, bioavailability,
stability or otherwise alter the properties of the nucleoside. A
number of nucleotide prodrug ligands are known. In general,
alkylation, acylation or other lipophilic modification of the mono,
di or triphosphate of the nucleoside will increase the stability of
the nucleotide. Examples of substituent groups that can replace one
or more hydrogens on the phosphate moiety are alkyl, aryl,
steroids, carbohydrates, including sugars, 1,2-diacylglycerol and
alcohols. Many are described in R. Jones and N. Bischofberger,
Antiviral Research, 27 (1995) 1-17. Any of these can be used in
combination with the disclosed nucleosides to achieve a desired
effect.
In one embodiment, the 1,3-oxaselenolanyl nucleoside is provided as
5'-hydroxyl lipophilic prodrug. Nonlimiting examples of U.S.
patents that disclose suitable lipophilic substituents that can be
covalently incorporated into the nucleoside, preferably at the
5'--OH position of the nucleoside or lipophilic preparations,
include U.S. Pat. No. 5,149,794 (Sep. 22, 1992, Yatvin, et al.);
U.S. Pat. No. 5,194,654 (Mar. 16, 1993, Hostetler, et al.); U.S.
Pat. No. 5,223,263 (Jun. 29, 1993, Hostetler, et al.); U.S. Pat.
No. 5,256,641 (Oct. 26, 1993, Yatvin, et al.); U.S. Pat. No.
5,411,947 (May 2, 1995, Hostetler, et al.); U.S. Pat. No. 5,463,092
(Oct. 31, 1995, Hostetler, et al.); U.S. Pat. No. 5,543,389 (Aug.
6, 1996, Yatvin, et al.); U.S. Pat. No. 5,543,390 (Aug. 6, 1996,
Yatvin, et al.); U.S. Pat. No. 5,543,391 (Aug. 6, 1996, Yatvin, et
al.); and U.S. Pat. No. 5,554,728 (Sep. 10, 1996, Basava, et al.),
all of which are incorporated herein by reference.
Foreign patent applications that disclose lipophilic substituents
that can be attached to the 1,3-oxaselenolanyl nucleosides of the
present invention, or lipophilic preparations, include WO 89/02733,
WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273,
WO/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.
Additional nonlimiting examples of derivatives of
1,3-oxaselenolanyl nucleosides are those that contain substituents
as described in the following publications. These derivatized
1,3-oxaselenolanyl nucleosides can be used for the indications
described in the text or otherwise as antiviral agents, including
as anti-HIV or anti-HBV agents. Ho, D. H. W. (1973) Distribution of
Kinase and deaminase of 1.beta.-D-arabinofuranosylcytosine in
tissues of man and mouse. Cancer Res. 33, 2816-2820; Holy, A.
(1993) Isopolar phosphorous-modified nucleotide analogues. In: De
Clercq (ed.), Advances in Antiviral Drug Design, Vol. I, JAI Press,
pp. 179-231; Hong, C. I., Nechaev, A., and West, C. R. (1979a)
Synthesis and antitumor activity of
1.beta.-3-arabinofuranosylcytosine conjugates of cortisol and
cortisone. Biochem. Biophys. Rs. Commun. 88, 1223-1229; Hong, C.
I., Nechaev, A., Kirisits, A. J. Buchheit, D. J. and West, C. R.
(1980) Nucleoside conjugates as potential antitumor agents. 3.
Synthesis and antitumor activity of
1-(.beta.-D-arabinofuranosyl)cytosine conjugates of corticosteriods
and selected lipophilic alcohols. J. Med. Chem. 28, 171-177;
Hostetler, K. Y., Stuhmiller, L. M., Lenting, H. B. M. van den
Bosch, H. and Richman, D. D. (1990) Synthesis and antiretroviral
activity of phospholipid analogs of azidothymidine and other
antiviral nucleosides. J. Biol. Chem. 266, 11714-11717; Hostetler,
K. Y., Korba, B. Sridhar, C., Gardener, M. (1994a) Antiviral
activity of phosphatidyl-dideoxycytidine in hepatitis B-infected
cells and enhanced hepatic uptake in mice. Antiviral Res. 24,
59-67; Hostetler, K. Y., Richman, D. D., Sridhar, C. N. Felgner, P.
L., Felgner, J., Ricci, J., Gerdener, M. F. Selleseth, D. W. and
Ellis, M. N. (1994b) Phosphatidylazidothymidine and
phosphatidyl-ddc: Assessment of uptake in mouse lymphoid tissues
and antiviral activities in human imnmunodeficiency virus-infected
cells and in rauscher leukemia virus-infected mice. Antimicrobial
Agents Chemother. 38, 2792-2797; Hunston, R. N., Jones, A. A.
McGuigan, C., Walker, R. T., Balzarini, J., and De Clercq, E.
(1984) Synthesis and biological properties of some cyclic
phosphotriesters derived from 2'-deoxy-5-fluorouridine. J. Med.
Chem. 27,440-444; Ji, Y. H., Moog, C., Schmitt, G., Bischoff, P.
and Luu, B. (1990); Monophosphoric acid diesters of
7.beta.-hydroxycholesterol and of pyrimidine nucleosides as
potential antitumor agents; synthesis and preliminary evaluation of
antitumor activity. J. Med. Chem. 33, 2264-2270; Jones, A. S.,
McGuigan, C., Walter, R. T., Balzarini, J. and DeClercq, E. (1984)
Synthesis, properties, and biological activity of some nucleoside
cyclic phosphoramidates. J. Chem. Soc. Perkin Trans. I, 1471-1474;
Juodka, B. A. and Smart, J. (1974) Synthesis of ditribonucleoside a
(P.fwdarw.N) amino acid derivatives. Coll. Czech. Chem. Comm. 39,
363-968; Kataoka, S., Imai, J., Yamaji, N., Kato, M., Saito, M.,
Kawada, T. and Imai, S. (1989) Alkylacted cAMP derivatives;
selective synthesis and biological activities. Nucleic Acids Res.
Sym. Ser., 21, 1-2; Kataoka, S., Uchida, R. and Yamaji, N. (1991) A
convenient synthesis of adenosine 3',5' cyclic phosphate (cAMP)
benzyl and methyl triesters. Heterocycles 32, 1351-1356;
Kinchington, D., Harvey, J. J., O'Connor, T. J., Jones, B. C. N.
M., Devine, K. G., Taylor-Robinson, D., Jeffries, D. J. and
McGuigan, C. (1992) Comparison of antiviral effects of zidovudine
phosphoramidate and phosphorodiamidate derivatives against HIV and
ULV in vitro. Antiviral Chem. Chemother. 3, 107-112; Kodama, K.,
Morozumi, M., Saitoh, K. I., Kuninaka, H., Yoshino, H. and
Saneyoshi, M. (1989) Antitumor activity and pharmacology of
1-.beta.-D-arabinofuranosylcytosine-5'-stearylphosphate; an orally
active derivative of 1-.beta.-D-arabinofuranosylcytosine. Jpn. J.
Cancer Res. 80, 679-685; Korty, M. and Engels, J. (1979) The
effects of adenosine- and guanosine 3',5'-phosphoric and acid
benzyl esters on guinea-pig ventricular myocardium.
Naunyn-Schmiedeberg's Arch. Pharmacol. 310, 103-111; Kumar, A.,
Goe, P. L., Jones, A. S. Walker, R. T. Balzarini, J. and De Clercq,
E. (1990) Synthesis and biological evaluation of some cyclic
phosphoramidate nucleoside derivatives. J. Med. Chem. 33,
2368-2375; LeBec, C., and Huynh-dinh, T. (1991) Synthesis of
lipophilic phosphate triester derivatives of 5-fluorouridine and
arabinocytidine as anticancer prodrugs. Tetrahedron Lett. 32,
6553-6556; Lichtenstein, J., Barner, H. D. and Cohen S. S. (1960)
The metabolism of exogenously supplied nucleotides by Escherichia
coli., J. Biol. Chem. 235, 457-465; Lucthy, J., Von Daeniken, A.,
Friederich, J. Manthey, B., Zweifel, J., Schlatter, C. and Benn, M.
H. (1981) Synthesis and toxicological properties of three naturally
occurring cyanoepithioalkanes. Mitt. Geg. Lebensmittelunters. Hyg.
72, 131-133 (Chem. Abstr. 95, 127093); McGuigan, C. Tollerfield, S.
M. and Riley, P. A. (1989) Synthesis and biological evaluation of
some phosphate triester derivatives of the anti-viral drug Ara.
Nucleic Acids Res. 17, 6065-6075; McGuigan, C., Devine, K. G.,
O'Connor, T. J., Galpin, S. A., Jeffries, D. J. and Kinchington, D.
(1 990a) Synthesis and evaluation of some novel phosphoramidate
derivatives of 3'-azido-3'-deoxythymidine (AZT) as anti-HIV
compounds. Antiviral Chem. Chemother. 1, 107-113; McGuigan, C.,
O'Connor, T. J., Nicholls, S. R. Nickson, C. and Kinchington, D.
(1990b) Synthesis and anti-HIV activity of some novel substituted
dialkyl phosphate derivatives of AZT and ddcyd. Antiviral Chem.
Chemother. 1, 355-360; McGuigan, C., Nicholls, S. R., O'Connor, T.
J., and Kinchington, D. (1990c) Synthesis of some novel dialkyl
phosphate derivative of 3'-modified nucleosides as potential
anti-AIDS drugs. Antiviral Chem. Chemother. 1, 25-33; McGuigan, C.,
Devine, K. G., O'Connor, T. J., and Kinchington, D. (1991)
Synthesis and anti-HIV activity of some haloalky phosphoramidate
derivatives of 3'-azido-3'deoxythylmidine (AZT); potent activity of
the trichloroethyl methoxyalaninyl compound. Antiviral Res. 15,
255-263; McGuigan, C., Pathirana, R. N., Mahmood, N., Devine, K. G.
and Hay, A. J. (1992) Aryl phosphate derivatives of AZT retain
activity against HIV1 in cell lines which are resistant to the
action of AZT. Antiviral Res. 17, 311-321; McGuigan, C., Pathirana,
R. N., Choi, S. M., Kinchington, D. and O'Connor,
T. J. (1993a) Phosphoramidate derivatives of AZT as inhibitors of
HIV; studies on the caroxyl terminus. Antiviral Chem. Chemother. 4,
97-101; McGuigan, C., Pathirana, R. N., Balzarini, J. and De
Clercq, E. (1993b) Intracellular delivery of bioactive AZT
nucleotides by aryl phosphate derivatives of AZT. J. Med. Chem. 36,
1048-1052.
Alkyl hydrogen phophonate derivatives of the anti-HIV agent AZT may
be less toxic than the parent nucleoside analogue. Antiviral Chem.
Chemother. 5, 271-277; Meyer, R. B., Jr., Shuman, D. A. and Robins,
R. K. (1973) Synthesis of purine nucleoside 3',5'-cyclic
phosphoramidates. Tetrahedron Lett. 269-272; Nagyvary, J. Gohil, R.
N., Kirchner, C. R. and Stevens, J. D. (1973) Studies on neutral
esters of cyclic AMP, Biochem. Biophys. Res. Commun. 55, 1072-1077;
Namane, A. Goyette, C., Fillion, M. P., Fillion, G. and Huynh-Dinh,
T. (1992) Improved brain delivery of AZT using a glycosyl
phosphotriester prodrug. J. Med. Chem. 35, 3939-3044; Nargeot, J.
Nerbonne, J. M. Engels, J. and Leser, H. A. (1983) Natl. Acad. Sci.
U.S.A. 80, 2395-2399; Nelson, K. A., Bentrude, W. G., Stser, W. N.
and Hutchinson, J. P. (1987) The question of chair-twist equilibria
for the phosphate rings of nucleoside cyclic 3',5'-monophosphates.
.sup.1 HNMR and x-ray crystallographic study of the diasteromers of
thymidine phenyl cyclic 3',5'-monophosphate. J. Am. Chem. Soc. 109,
4058-4064; Nerbonne, J. M., Richard, S., Nargeot, J. and Lester, H.
A. (1984) New photoactivatable cyclic nucleotides produce
intracellular jumps in cyclic AMP and cyclic GMP concentrations.
Nature 301, 74-76; Neumann, J. M., Herve, M., Debouzy, J. C.,
Guerra, F. I., Gouyette, C., Dupraz, B. and Huynh-Dinh, T. (1989)
Synthesis and transmembrane transport studies by NMR of a glucosyl
phospholipid of thymidine. J. Am. Chem. Soc. 11 1, 4270-4277; Ohno,
R., Tatsumi, N., Hirano, M., Imai, K. Mizoguchi, H., Nakamura, T.,
Kosaka, M., Takatuski, K., Yamaya, T., Toyama, K., Yoshida, T.,
Masaoka, T., Hashimoto, S., Ohshima, T., Kimura, I., Yamada, K. and
Kimura, J. (1991) Treatment of myelodyspastic syndromes with orally
administered
1-.beta.-D-rabinofaranosylcytosine-5'-stearylphosphate. Oncology
48, 451-455. Palomino, E., Kessle, D. and Horwitz, J. P. (1989) A
dihydropyridine carrier system for sustained delivery of
2',3'dideoxynucleosides to the brain. J. Med. Chem. 32, 622-625;
Perkins, R. M., Barney, S., Wittrock, R., Clark, P. H., Levin, R.
Lambert, D. M., Petteway, S. R., Serafinowska, H. T., Bailey, S.
M., Jackson, S., Harnden, M. R., Ashton, R., Sutton, D., Harvey, J.
J. and Brown, A. G. (1993) Activity of BRL47923 and its oral
prodrug, SB203657A against a rauscher murine leukemia virus
infection in mice. Antiviral Res. 20 (Suppl. I). 84; Piantadosi,
C., Marasco, C. J., Jr., Morris-Natschke, S. L., Meyer, K. L.,
Gumus, F., Surles, J. R., Ishaq, K. S., Kucera, L. S. Iyer, N.,
Wallen, C. A., Piantadosi, S. and Modest, E. J. (1991) Synthesis
and evaluation of novel ether lipid nucleoside conjugates for
anti-HIV-1 activity. J. Med. Chem. 34, 1408-1414; Pompon, A.,
Lefebvre, I., Imbach, J. L., Kahn, S. and Farquhar, D. (1994)
Decomposition pathways of the mono- and bis(pivaloyloxymethyl)
esters of azidothymidine-5'-monophosphate in cell extract and in
tissue culture medium; an application of the on-line ISRP-cleaning'
HPLC technique. Antiviral Chem. Chemother. 5, 91-98; Postemark, T.
(1974) Cyclic AMP and cyclic GMP. Anu. Rev. Pharmacol. 14, 23-33;
Prisbe, E. J., Martin, J. C. M., McGee, D. P. C., Barker, M. F.,
Smee, D. F. Duke, A. E., Matthews, T. R. and Verheyden, J. P. J.
(1986) Synthesis and antiherpes virus activity of phosphate and
phosphonate derivatives of 9-[(1,3-dihydroxy-2-propoxy)methyl]
guanine. J. Med. Chem. 29, 671-675; Pucch, F., Gosselin, G.,
Lefebvre, I., Pompon, A., Aubertin, A. M. Dim, A. and Imbach, J. L.
(1993) Intracellular delivery of nucleoside monophosphate through a
reductase-mediated activation process. Antiviral Res. 22, 155-174;
Pugaeva, V. P., Kochkeva, S. I., Mashbits, F. D. and Eizengart, R.
S. (1969). Toxicological assessment and health standard ratings for
ethylene sulfide in the industrial atmosphere. Gig. Trf. Prof.
Zabol. 13, 47-48 (Chem. Abstr. 72, 212); Robins, R. K. (1984) The
potential of nucleotide analogs as inhibitors of retroviruses and
tumors. Pharm. Res. 11-18; Rosowsky, A., Kim, S. H., Ross and J.
Wick, M. M. (1982) Lipophilic 5'-(alkylphosphate) esters of
1-.beta.-D-arabinofuranosylcytosine and its N.sup.4 -acyl and
2.2'-anhydro-3'0-acyl derivatives as potential prodrugs. J. Med.
Chem. 25, 171-178; Ross, W. (1961) Increased sensitivity of the
walker turnout towards aromatic nitrogen mustards carrying basic
side chains following glucose pretreatment. Biochem. Pharm. 8,
235-240; Ryu, E. K., Ross, R. J., Matsushita, T., MacCoss, M.,
Hong, C. I. and West, C. R. (1982). Phospholipid-nucleoside
conjugates 3. Synthesis and preliminary biological evaluation of
1-.beta.-D-arabinofuranosylcytosine 5'diphosphate[-],
2-diacylglycerols. J. Med. Chem. 25, 1322-1329; Saffhill, R. and
Hume, W. J. (1986) The degradation of 5-iododeoxyurindine and
5-bromoeoxyuridine by serum from different sources and its
consequences for the use of these compounds for incorporation into
DNA. Chem. Biol. Interact. 57, 347-355; Saneyoshi, M., Morozumi,
M., Kodama, K., Machida, J., Kuninaka, A. and Yoshino, H. (1980)
Synthetic nucleosides and nucleotides XVI. Synthesis and biological
evaluations of a-series of 1-.beta.-D-arabinofuranosylcytosine
5'-alkyl or arylphosphates. Chem. Pharm. Bull. 28, 2915-2923;
Sastry, J. K., Nehete, P. N., Khan, S., Nowak, B. J., Plunkett, W.,
Arlinghaus, R. B. and Farquhar, D. (1992) Membrane-permeable
dideoxyuridine 5'-monophosphate analogue inhibits human
immunodeficiency virus infection. Mol. Pharmacol. 41, 441-445;
Shaw, J. P., Jones, R. J. Arimilli, M. N., Louie, M. S., Lee, W. A.
and Cundy, K. C. (1994) Oral bioavailability of PMEA from PMEA
prodrugs in male Sprague-Dawley rats. 9th Annual AAPS Meeting. San
Diego, CA (Abstract). Shuto, S., Ueda, S., Imamura, S., Fukukuawa,
K. Matsuda, A. and Ueda, T. (1987) A facile one-step synthesis of
5'-phosphatidylnucleosides by an enzymatic two-phase reaction.
Tetrahedron Lett. 28, 199-202; Shuto, S., Itoh, H., Ueda, S.,
Imamura, S., Kukukawa, K., Tsujino, M. Matsuda, A. and Ueda, T.
(1988) A facile enzymatic synthesis of
5'-(3-sn-phosphatidyl)nucleosides and their antileukemic
activities. Chem. Pharm. Bull. 36, 209-217. One preferred phosphate
prodrug group is the S-acyl-2-thioethyl group, also referred to as
"SATE."
II. Preparation of the Active Compounds
1,3-Oxaselanolanyl nucleosides have evaded production to date
because of difficulties encountered with construction of the
1,3-oxaselenolane ring. A process for the production of this ring
is now provided herein. One embodiment of the process is
illustrated in FIG. 1.
Processes are also provided for the preparation of isolated
.beta.-D (i.e., 2S,5R) and .beta.-L1,3oxaselenolanyl (i.e., 2R,5S)
nucleosides. One exemplification of this process is illustrated in
FIG. 2. The numbering scheme for the compounds used in the Examples
below is provided in FIG. 1.
EXAMPLE 1
Preparation of 1,3-oxaselenolane Ring
Selenocyanate was prepared by the method of Kirby in excellent
yield. In the first step, ethylbromoacetate (BrCH.sub.2 CO.sub.2
Et) is reacted with potassium selenyl acetate in alcohol to form
selenocyanate 2.
In order to construct lactone 5, it was initially attempted to
reduce the selenocyanate 2 with NaBH.sub.4 and hydrolyze the
resulting ester with aqueous NaOH to the selenol acetic acid, which
could be used for the construction of the oxaselenolane ring system
5. However, selenol acetic acid decomposed during the acidification
with HCl at pH2. It has been reported that selenols can be readily
oxidized by oxygen in air to stable dimers which can be reduced
back to selenols by H.sub.3 PO.sub.2. It was discovered that the
reduction of the bis(selenoacetic acid) to selenol as well as
cyclization can take place in a one-pot reaction without isolation
of the intermediates. Thus, dimer 3 was prepared in 81% yield by
refluxing 1 with KSeCN in ethanol for 1 hour followed by reduction
with NaBH.sub.4 at 0.degree. C. for 20-30 minutes. Compared to the
recently reported procedure for the preparation of diselenides,
this method has the advantages of milder reaction conditions, a
high yield and an easier workup. Lactone 5 was then prepared in 33%
yield by hydrolysis of 3 with refluxing aqueous acetic acid (50%)
for 24 hours followed by the reduction to selenol acetic acid with
H.sub.3 PO.sub.2 which was condensed in situ with
2-benzoyloxyacetaldehyde in the presence of H.sub.3 PO.sub.2 under
nitrogen. For reduction of the lactone 5, it was found that DIBAL-H
can selectively reduce the lactone over the ester in THF, while no
selectivity was observed in toluene. Thus, sugar acetate 7 was
prepared by DIBAL-H reduction of 5 in THF followed by in situ
acetylation with acetic anhydride. Condensation of the acetate 7,
without purification, with silylated bases in the presence of
SnCl.sub.4 or TMSOTf gave inseparable mixtures of .alpha.- and
.beta.-isomers 8a and 8b. Removal of the benzoyl protecting group
of 8a and 8b by methylamine or ammonia in methanol the final
nucleosides as an .alpha./.beta.-mixture. The .alpha.-cytosine
nucleoside was obtained by repeated recrystallization of the
.alpha./.beta.-mixture from MeOH/Et.sub.2 O and then methanol,
while .beta.-cytosine nucleoside (9a) was obtained by HPLC
separation of the mother liquor (C.sub.18 -Column, 20% MeOH in
H.sub.2 O). The .beta. and .alpha.-5-fluoro-cytosine nucleosides
were obtained by silica gel chromatographic separation of the
.alpha./.beta.-mixture. The structures of the synthesized
selenolane nucleosides were confirmed by elemental analyses, .sup.1
H and .sup.13 C NMR. Stereochemical assignments were determined
based on 2D-NOESY experiments in which a correlation between 2'--H
and 5'--H of .beta.-isomer 9b was observed while an absence of this
correlation in .alpha.-isomer 10b was noted. The assignment of
stereochemistry was also supported by the upfield chemical shifts
of 2'--H in 9a and 9b compared to that of 10a and 10b due to
deshielding by the heterocyclic bases.
Stereochemistry
Since the 1' and 4' carbons of the 1,3-oxaselenolanyl moiety of the
nucleoside is chiral, their nonhydrogen substituents (the
pyrimidine or purine base and the CHOR groups, respectively) can be
either cis (on the same side) or trans (on opposite sides) with
respect to the sugar ring system. The four optical isomers
therefore are represented by the following configurations (when
orienting the sugar moiety in a horizontal plane such that the
oxygen atom is in the back): cis (with both groups "up", which
corresponds to the configuration of naturally occurring
nucleosides), cis (with both groups "down", which is a nonnaturally
occurring configuration), trans (with the C2' substituent "up" and
the C4.varies.0 substituent "down"), and trans (with the C2'
substituent "down" and the C4' substituent "up"). The
"D-nucleosides" are cis nucleosides in a natural configuration and
the "L-nucleosides" are cis nucleosides in the nonnaturally
occurring configuration.
The enantiomers of 1,3-oxaselenolanyl nucleoside were obtained in
two ways; by chiral chromatography of the nucleoside as described
in Example 2 and by fractional crystallization of L-menthol
disastereomers of 1,3-oxaselenolane followed by condensation of the
resolved 1,3-oxaselenolanyl nucleoside with the desired base in the
presence of a Lewis acid that doesn't racemize the oxaselenolane
ring.
EXAMPLE 2
Resolution of .beta.-D and .beta.-L Enantiomers of
2-hydroxymethyl-4-(n-5'-cytosin-1'-yl)-1,3-oxaselenolane and
2-hydroxymethyl-4-(n-5'-fluorocytosin-1'-yl)-1,3-oxaselenolane by
chiral chromatography
2-Hydroxymethyl-4-(N-5'-cytosin-1'-yl)-1,3-oxaselenolane and
2-hydroxymethyl-4-(N-5'-flurocytosin-1'-yl)-1,3-oxaselenolane were
resolved by chiral chromatography. The compound (racemic, ca. 2 mg)
was dissolved in a minimum amount (ca. 400 .mu.L) of methanol (HPLC
grade). The following conditions were used for the resolution:
Waters HPLC system; Column: Chiralpak AS 4.6.times.250 mm; Mobile
phase: 2-propanol, Flow rate: 0.80 mL/min; Detector: UV-260 nm;
Sparge gas; Helium; Sparge speed: 25 mL/min/solvent reservoir;
Injection amount: 20 .mu.L of the solution each time; Retention
times; (-)-(2S,5R)-.beta.-L-2',3'-dideoxy-3'-seleno-cytidine, 5.50
min; (+)-(2R,5S)-.beta.-D-2',3'-dideoxy-3'-seleno-cytidine, 6.92
min;
(-)-(2S,5R)-.beta.-L-2',3'-dideoxy-5-fluoro-3'-seleno-cytidine,
5.97 min;
(+)-(2R,5S)-.beta.-D-2',3'-dideoxy-5-fluoro-3'-seleno-cytidine,
9.62 min. The optical purities of the resolved compounds were
>95% ee.
EXAMPLE 3
Resolution of .beta.-D and .beta.-L Enantiomers of
1,3-oxaselenolanyl Intermediates by Conversion to Diastereomers
Followed by Separation of Diastereomers by Fractional
Crystallization
(-)-L-Mentholcarboxyal. To a mixture (-)-L-menthol (30 g, 0.2 mol)
and gluoxylic acid (36.8 g, 0.4 mol) in toluene (1000 ml) p-TsOH (5
g) was added and the reaction mixture was stirred at 100 C for 3
hours. When the reaction finished p-TsOH was neutralized with
Et.sub.3 N and evaporated to dryness. The residue was dissolved in
CHCl.sub.3 (500 ml), washed with water (3.times.500 ml), the
organic layer was collected, dried (Na.sub.2 SO.sub.4) and
evaporated. The oil was crystallized from petroleum either to give
(-)-L-mentholcarboxyal as white crystals 20 g (50%): mp 82.degree.
C.; .sup.1 H NMR (CHCl.sub.3) .delta. 9.40 (s, 1H, CHO), 4.78 (dt,
J=4.45, 11 Hz, 1H, 1-H), 0.75-2.03 (m, 19H); .sup.13 C NMR
(CHCl.sub.3) .delta. 184.41, 170.22, 87.13, 46.79, 40.40, 34.00,
31.42, 26.11, 23.28, 21.94, 20.68, 16.15. Anal. Calcd for C.sub.12
H.sub.20 O.sub.3 : C, 67.89; H, 9.50; Found: C, 67.65; H, 9.67. M/S
m/e 212.3 (M+).
[2-(1'R,2'S,5'R)-Menthyl-(5-one-1,3-oxaselnolane)]-L-carboxylate
(11) and
[2-(1'R,2'S,5'R)-Menthyl-(5-one-1,3-oxaselenolane-)]-D-carboxylate.
To a solution (-)-L-mentholcarboxyal (6.4 g, 30 mmol) in toluene
(100 ml) (SeCH.sub.2 COOH).sub.2 (4.15 g, 15 mmol) was added and
reaction mixture was gently heated to 100.degree. C. under argon
atmosphere with stirring. Hypophosphorous acid (50% water solution,
2.7 ml) was added dropwise for one hour. The reaction mixture was
then refluxed additionally for one hour with vigorous stirring
under argon atmosphere. The reaction mixture was evaporated to 20
ml, diluted with EtOAc (250 ml), and washed with water (3.times.500
ml). The organic layer was collected, dried (Na.sub.2 SO.sub.4) and
evaporated. The residue was purified by column chromatography over
SiO.sub.2 using the mixture EtOAc-Hex (1:10, V/V) as eluent, to
give 11 as a solid 3.9 g (77.6%). Crystallization of the mixture
compounds from hexanes at room temperature gave 11 as fine
colorness needles: mp 106.5.degree. C.; [.alpha.].sup.25.sub.D
=59.86.degree. (c 0.5, CHCl.sub.3); .sup.1 H NMR (CHCl.sub.3)
.delta. 5.83 (s, 1H, 2'-H), 4.77 (dt, J=4.45, 12 Hz, 1H, 1-H), 3.97
(d, J=15.34 Hz, 1H, 4'-H.sub.b), 3.67 (dt, J=15.35 Hz, .sup.4
J=21.17 Hz, 1H, 4'-H.sub.a), 0.75-2.03 (m, 19H); .sup.13 C NMR
(CHCl.sub.3) .delta. 173.97, 168.67, 76.88, 63.84, 47.07, 40.46,
34.02, 31.38, 26.07, 23.23, 22.65, 21.93, 20.71, 16.11. Anal. Calcd
for C.sub.14 H.sub.22 O.sub.4 Se: C, 50.45; H, 6.65; Found: C,
50.65; H, 6.62 MS m/e 333 (M+). Crystallization mother liquid at
-5.degree. C.; [.alpha.].sup.25.sub.D =-111.71.degree. (c 0.5,
CHCl.sub.3); .sup.1 H NMR (CHCl.sub.3) .delta. 5.83 (s, 1H, 2'-H),
4.78 (dt, J=4.45, 12 Hz, 1H, 1-H), 3.95 (d, J=15.41 Hz, 1H,
4'-H.sub.a), 3.68 (dt, J=15.45 Hz, .sup.4 J=19.35 Hz, 1H,
4'-H.sub.a), 0.75-2.03 (m, 19H); .sup.13 C NMR (CHCl.sub.3) .delta.
173.98, 168.63, 76.15, 63.76, 46.95, 39.88, 34.01, 31.32, 26.22,
23.24, 22.98, 21.94, 20.74, 16.14; Anal. Calcd for C.sub.14
H.sub.22 O.sub.4 Se: C, 50.45; H, 6.65; Found: C, 50.47; H, 6.63
M/S m/e 333 (M+).
1-.beta.-L-(2'-Hydroxymethyl-1,3'-oxaselenolan-5'yl)-5-fluorocytosine
(15) and
1-.alpha.-L-(2'-hydroxymethyl-1',3'-oxaselnolane-5'-yl)-5-fluorocytosine
(16).
To a solution of lithium tri-tert-butoxyaluminohydride (6 mmol, 6
ml 1M solution in the THF) of the solution lactone 11 (1 g, 3.33
mmol) in the 5 ml THF was added dropwise at -10.degree. C. for one
hour with stirring under argon atmosphere. Then acetic anhydride (2
g, 20 mmol) was added slowly with stirring at -5-0.degree. C. The
reaction mixture was stirred additionally for one hour, diluted
with EtOAc (100 ml), washed with water (3.times.100 ml), dried
(Na.sub.2 SO.sub.4), and concentrated to dryness to give a crude
5'-acetate 13. The sugar acetate 13 was dissolved in CH.sub.2
CI.sub.2 (5 ml) and slowly added to silylated 5-flurocytosine
prepared by stirring of the mixture 5-fluorocytosine (0.34 g, 2.63
mmol), 2,4,6-collidine (0.8 ml, 6.61 mmol) and,
tert-butyldimethylsilyl
trifuloromethanesulfonate (1.32 g, 5.08 mmol) for one hour under
argon atmosphere. To the resulting mixture was added
iodotrimethylsilane (0.35 g, 1.75 mmol), stirred at room
temperature for 18 hours, diluted with CHCl.sub.3 (100 ml), poured
into aq. Na.sub.2 S.sub.2 O.sub.3 (100 ml), washed with water,
dried (Na.sub.2 SO.sub.4), and concentrated to dryness. The residue
was purified by flash-chromatography over silica gel using
CHCl.sub.3 as eluent to give crude 13 as solid (0.15 g, 11.2%).
.sup.1 H NMR (CDCl.sub.3) .delta. 8.35 (d, J=6.3 Hz, 1H, 6-H),
7.55, 7.53 (2.times.br s, 2H, NH.sub.2), 6.45 (m, 1-h, 5'-H), 6.14
(m, 1H, 2'-H), 4,79 (m, 1H, 1-H), 3.66 (m, 2H, 6'-H.sub.ab). A
solution of the compound 14 (0.15 g, 0.33 mmol) in THF (10 ml) at
room temperature under argon for one hour. The reaction mixture was
stirred additionally 1 hour, quenched with MeOH (5 ml) and
resulting mixture was applied to short column with silica gel. The
column was eluted with mixture EtOAc-Hex-MeOH (1:1:1, V/V, 100 ml).
The eluent was concentrated to dryness and resulting solid purified
over SiO.sub.2 using CHCl.sub.3 -EtOH (20:1, V/V) as eluent to give
mixture .beta.-L- (15) and .alpha.-L-nucleosides (16) like white
solid 0.033 g (34%). The mixture was reseparated by column over
SiO.sub.2 using as eluent mixture four solvents
EtOAc-Hex-CHCl.sub.3 -EtOH (5:5:2:1, V/V).
1-.beta.-L-(2'-Hydroxymethyl-1',3'-oxaselenolane-5'-yl)-5-fluorocytosine
(15).
White solid (0.01 g, 10.2%); mp 186-189.degree. C. (MeOh);
[.alpha.].sup.25.sub.D =-55.69.degree. (c 0.35, MEOH); UV
(H.sub.2)) .lambda..sub.max 280.0 nm (.epsilon. 10646, pH2), 280.0
nm (.epsilon. 7764, pH 11); .sup.1 H NMR (DMSO-d.sub.6) .delta.
8.07 (d, J=7.1 Hz, 1H, 6-H), 7.92, 7.67 (2.times.br s, 2H,
NH.sub.2, D.sub.2 O exchangeable), 6.06 (t, J=2.96 Hz, 1-H, 5'-H),
5.42 (t, J=4.82 Hz, 1H, 2'-H), 5.34 (t, J=5.68 Hz, 1H, OH, D.sub.2
O exchangeable), 3.81 (m, 1H, 6'-H.sub.a), 3.68 (m, 1H,
6'-H.sub.b), 3.39 (dd, J=4.84 Hz, 1H, 4'-H.sub.b), 3.08 (dd, J=8.11
Hz, 1H, 4'-H.sub.a); .sup.13 C NMR (DMSO-d.sub.6) .delta. 157.7
(C.dbd.O), 153.3 (4-C), 137.6 (6-C), 135.2 (5-C), 88.3 (5'-C), 78.2
(2'-C), 64.0 (6'-C), 28.9 (4'-C); Anal. Calcd for C.sub.8 H.sub.10
O.sub.3 N.sub.3 FSe: C, 32.67, H, 3.43, N, 14.29; Found: C, 32.62;
H. 3.51, N, 14.41; M/S m/e 295 (M+).
1'-.alpha.-L-(2'-Hydroxymethyl-1',3'-oxaselenolane-5'-yl)-5-flurocytosine
(16).
White solid (0.013 g, 13.2%); mp 193-195.degree. C. (MeOH);
[.alpha.].sup.25.sub.D =+84.20.degree. (c 0.26,MeOH); UV (H.sub.2
O) .lambda..sub.max 279.5 nm (.epsilon. 7638, pH 7), 287.5 nm
(.epsilon. 9015, pH 2), 281.0 nm (.epsilon. 6929, pH 11); .sup.1 H
NMR (DMSO-d.sub.6) .delta. 7.91 (d, J=7.1 Hz, 1H, 6-H), 7.88, 7.63
(2.times.br s, 2H, NH.sub.2, D.sub.2 O exchangeable), 6.35 (t,
J=4.95 Hz, 1-H, 5'-H), 5.63 (dd, J=4.83 Hz, 1H, 2'-H), 5.28 (t,
J=5.67 Hz, 1H, OH, D.sub.2 O exchangeable), 3.70 (m, 1H,
6'-H.sub.a), 3.53 (m, 1H, 6'-H.sub.b), 3.47 (dd, J=4.82 Hz, 1H,
4'-H.sub.a), 3.24 (dd, J=7.88 Hz, 1H, 4'-H.sub.b); .sup.13 C NMR
(DMSO-d.sub.6) .delta. 157.8 (C.dbd.O), 153.2 (4-C), 137.3 (6-C),
134.9 (5-C), 88.6 (5'-C), 80.9 (2'-c), 65.5 (6'-C), 29.4 (4'-C);
Anal. Calcd for C.sub.8 H.sub.10 O.sub.3 N.sub.3 FSe: C, 32.67, H,
3.43, N, 14.29; Found: C, 32.59; H, 3.49, N, 14.20; M/S m/e 295
(M+). Synthesis of nucleosides 8 and 9 has been accomplished in
same manner from a lactone 3 (1 g, 3.33 mmol) to give
1-.beta.-D-(2'-hydroxymethyl-1',3'-oxaselnolane-5'-yl)-5-fluorocytosine
8. White solid (0.007 g, 8.5%); mp 186-189.degree. C. (MeOH);
[.alpha.].sup.25.sub.D =+56.21.degree. (c 0.33, MeOH); UV (H.sub.2
O) Amax 280.0 nm (.epsilon. 8576, pH 7), 289.0 nm (.epsilon. 10456,
pH 2), 280.0 nm (.epsilon. 7795, pH 11): 1H NMR (DMSO-d.sub.6)
.delta. 8.07 (d, J=7.1 Hz, 1H, 6-H), 7.92, 7.67 (2.times.br s, 2H,
NH.sub.2, D.sub.2 O exchangeable), 6.06 (5, J=2.96 Hz, 1-H, 5'-H),
5.42 (5, J=4.82 Hz, 1-H, 2'-H), 5.34 (5, J=5.68 Hz, 1H, OH, D.sub.2
O exchangeable), 3.81 (m, 1H, 6'-H.sub.a), 3.68 (m, 1H,
6'-H.sub.b), 3.39 (dd, J=4.84 Hz, 1H, 4'-H.sub.b), 3.08 (dd, J=8.11
Hz, 1H, 4'-H.sub.a); .sup.13 C NMR (DMSO-d.sub.6 (.delta. 157.7
(C.dbd.O), 153.3 (4-C), 137.6 (6-C), 135.2 (5-C), 88.3 (5'-C), 78.2
(2'-C), 64.0 (6'-C), 28.9 (4'-C); Anal. Calcd for C.sub.8 H.sub.10
O.sub.3 N.sub.3 FSe: C, 32.67, H, 3.43, N, 14.29; Found: C, 32.57;
H, 3.39, N, 14.35; M/S m/e 295 (M+).
1-.alpha.-D-(2'-Hydroxymethyl-1',3'-oxaselenolan-5'-yl)-5-fluorocytosine
9.
White solid (0.01 g, 10%); mp 193-195.degree. C. (MeOH);
[.alpha.].sup.25.sub.D =-85.49.degree.(c 0.3 1, MeOH); UV (H.sub.2
O) .lambda..sub.max 279.5 nm (.epsilon. 7644, pH 7), 287.5 nm
(.epsilon. 9067, pH 2), 281.0 nm (.epsilon. 6983, Ph 11); .sup.1 H
NMR (DMSO-d.sub.6) .delta. 7.91 (d, J=7.1 Hz, 1H, 6-H), 7.88, 7.63
(2.times.br s, SH, NH.sub.2, D.sub.2 O exchangeable), 6.35 (5,
J=4.95 Hz, 1-H, 5'-H), 5.63 (dd, J=4.83 Hz, 1H, 2'-H), 5.28 (5,
J=5.67 Hz, 1H, OH, D.sub.2 O exchangeable), 3.70 (m, 1H,
6'-H.sub.a); .sup.13 C NMR (DMSO-d.sub.6) .delta. 157.8 (C.dbd.O),
153.2 (4-C), 137.3 (6-C), 134.9 (5-C), 88.6 (5'-C), 80.9 (2'-C),
65.5 (6,-C), 29.4 (4'-C); Anal. Calcd for C.sub.8 H.sub.10 O.sub.3
N.sub.3 FSe: C, 32.67, H, 3.43, N. 14,29; Found: C, 32.67; H, 3.48;
N, 14.47; M/S m/e 295 (M+).
Table 1 provides the separation results for (+)-.beta.-Se-FddC,
(-)-.beta.-Se-FddC, (+)-.alpha.-Se-FddC, (-)-(.alpha.-Se-FddC and
(-)-.beta.-Se-ddC and compares the retention times and absorption
wavelengths of these compounds with (-)-.beta.-FTC and
(+)-.beta.-FTC.
TABLE 1 ______________________________________ Separation results
Optical Retention Absorption rotation Compounds time (min)
wavelength, nm (degrees) Purity
______________________________________ (-)-.beta.-Se-FddC 4.8
247.3, 285.1 -103.2 100 (c0.5, MeOH) (+)-.beta.-Se-FddC 7.7 247.3,
285.1 +96.8 100 (c).5, MeOH) (-)-.alpha.-Se-FddC 4.8 247.3, 285.1
ND 100 (+)-.alpha.-Se-FddC 6.6 247.3, 285.1 ND 90 (-)-.beta.-FTC
4.7 242.6, 285.1 -- -- (+)-.beta.-FTC 6.9 242.6, 285.1 -- --
(-)-.beta.-Se-ddC 9.5 242.6, 270.9 -72.4 100 (cl, DMSO)
(+)-.beta.-Se-ddC 11.9 242.6, 270.9 +56.4 96 (cl, DMSO)
(-)-.alpha.-Se-ddC ND 242.6, 270.9 -46.7 100 (cl, DMSO)
(+)-.alpha.-Se-ddC ND 242.6, 270.9 +26.1 94 (cl, DMSO)
______________________________________
Table 2 provides resolution and separation factors of compounds
separated on ChiralPak AS. The separation factor is defined as the
retention time of the second eluted isomer minus dead time per the
difference between retention time of the first eleuted isomer and
dead time. The resolution factor is defined as twice the difference
of retention time of (+) and (-) isomers per the band width of the
two peaks.
TABLE 2 ______________________________________ Comparison of
separations on Chiralpak AS. Chromatagraphic conditions: mobil
phase; 2-propanol; 100 .mu.g in 10 .mu.l of methanol were injected;
UV detection at 254 nm; Flow rate at ml/min. Separation Resolution
Compounds factor .alpha..sup.a R.sub.s.sup.b
______________________________________ racemic .alpha.-Se-FddC 2.34
1.91 racemic .beta.-Se-FddC 3.14 3.28 racemic .beta.-FTC 2.84 2.87
______________________________________ .sup.a Separation factor =
(retention time of the second eluted isomer dead time)/(retention
time of the first eluted isomer dead time). .sup.b Resolution
factor = 2 .times. [difference of retention time of (+) and (-)
isomers]/(the band width of the two peaks).
Table 3 gives the effects of various solvent rations and flow rate
chiral paration of racemic .beta.-Se-ddC.
TABLE 3 ______________________________________ The effects of
various solvent ratios and flow rate chiral paration of racemic
.beta.-Se-ddC First eluted EtOH: peak area First peak Hexane
Flow-rate Resolution (uV*sec .times. retention Ratio (ml/min)
R.sub.s 10.sup.7) time (min) ______________________________________
100:0 0.8 1.25 1.25 4.65 50:50 0.8 1.48 1.13 6.06 40:60 0.8 1.76
1.11 7.21 30:70 0.8 2.05 1.08 9.71 30:70 1.0 1.98 0.88 7.83 30:70
1.4 1.90 0.64 5.59 20:80 1.4 2.12 0.61 9.78 40:60 0.6 1.75 1.46
9.53 ______________________________________
Mono, di, and triphosphate derivatives of the active nucleosides
can be prepared as described according to published methods. The
monophosphate can be prepared according to the procedure of Imai,
et al., J. Org. Chem., 34(6), 1547-1550 (June 1969). The
diphosphate can be prepared according to the procedure of Davisson,
et al., J. Org. Chem., 52(9), 1794-1801 (1987). The triphosphate
can be prepared according to the procedure of Hoard, et al., J. Am.
Chem. Soc., 87(8), 1785-1788 (1965).
III. Combination and Alternation Therapies
It has been recognized that drug-resistant variants of HIV and HBV
can emerge after prolonged treatment with an antiviral agent. Drug
resistance most typically occurs by mutation of a gene that encodes
for an enzyme used in the viral lifecycle, and most typically in
the case of HIV, reverse transcriptase, protease, or DNA
polymerase, and in the case of HBV, DNA polymerase. Recently, it
has been demonstrated that the efficacy of a drug against HIV
infection can be prolonged, augmented, or restored by administering
the compound in combination or alternation with a second, and
perhaps third, antiviral compound that induces a different mutation
from that caused by the principle drug. Alternatively, the
pharmacokinetics, biodistribution, or other parameter of the drug
can be altered by such combination or alternation therapy. In
general, combination therapy is typically preferred over
alternation therapy because it induces multiple simultaneous
stresses on the virus.
The second antiviral agent for the treatment of HIV, in one
embodiment, can be a reverse transcriptase inhibitor (a "RTI"),
which can be either a synthetic nucleoside (a "NRTI") or a
non-nucleoside compound (a "NNRTI"). In an alternative embodiment,
in the case of HIV, the second (or third) antiviral agent can be a
protease inhibitor. In other embodiments, the second (or third)
compound can be a pyrophosphate analog, or a fusion binding
inhibitor. A list compiling resistance data of in vitro and in vivo
for a number of antiviral compounds is found in Schinazi, et al.,
"Mutations in retroviral genes associated with drug resistance,"
International Antiviral News, Volume 1(4), International Medical
Press 1996.
Preferred compounds for combination or alternation therapy for the
treatment of HBV include FTC (the (-)-enantiomer or the racemate),
L-FMAU, interferon, .beta.-D-dioxoxlanyl-guanine (DXG),
.beta.-D-dioxolanyl-2,6-diaminopurine (DAPD), and
.beta.-d-dioxolanyl-6-chloropurine (ACP), famciclovir, penciclovir,
BMS-200475, bis bom PMEA (adefovir, dipivoxil); lobucavir,
ganciclovir, and ribavarin.
Preferred examples of antiviral agents that can be used in
combination or alternation with the compounds disclosed herein for
HIV therapy include
2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC); the
(-)-enantiomer of 2-hydroxymethyl-5-(cyostin-1-yl)-1,3-oxathiolane
(3TC); carbovir, acyclovir, interferon, AZT, DDI, DDC, D4T, CS-92
(3'-azido-2',3-dideoxy-5-methyl-cytidine), and .beta.-D-dioxolane
nucleosides such as .beta.-D-dioxolanyl-guanine (DXG),
.beta.-D-dioxolanyl-6-chloropurine (ACP), and MKC-442
(6-benzyl-1-(ethoxymethyl)-5-isopropyl uracil.
Preferred protease inhibitors include crixovan (Merck), nelfinavir
(Agouron), ritonavir (Abbot), saquinavir (Roche), and DMP-450
(DuPont Merck).
Nonlimiting examples of compounds that can be administered in
combination or alternation with any of the 1,3-oxaselenolenyl
nucleosides include
(1S,4R)-4-[2-amino-6-cyclopropyl-amino)-9H-purin-9-yl]-2-cyclopentene-1-me
thanol succinate ("1592", a carbovir analog; Glaxo Wellcome); 3TC:
(-)-.beta.-L-2',3'-dideoxy-3'-thiacytidine (Glaxo Wellcome); a-APA
R18893: a-nitro-anilino-phenylacetamide; A-77003; C2 symmetry-based
protease inhibitor (Abbott); A-75925: C2 symmetry-based protease
inhibitor (Abbott); AAP-BHAP: bisheteroarylpiperazine analog
(Upjohn); ABT-538: C2 symmetry-based protease inhibitor (Abbott);
AzddU: 3'-azido-2',3'-dideoxyuridine; AZT:
3'-azido-3'-deoxythymidine (Glaxo Wellcome); AZT-p-ddI:
3'-azido-3'-deoxythymidilyl-(5',5')-2',3-'dideoxyinosinic acid
(Ivax): BHAP: bisheteroarylpiperazine; BILA 1906:
N-{1S-{{{3-[2S-{(1,1-dimethylethyl)amino]carbonyl}-4R-]3-pyridinylmethyl)t
hio]-1-piperidinyl]-2R-hydroxy-1S-(phenylmethyl)-propyl]amino]carbonyl]-2-m
ethylpropyl}-2-quinolinecarboxamide (Bio
Mega/Boehringer-Ingelheim); BILA 2185:
N-(1,1-dimethyllethyl)-1-[2S-[[2-2,6-dimethyphenoxy)-1-oxoethyl]amino]2-R-
hydroxy-4-phenylbutyl]4R-pyridinylthio-2-piperidinecarboxamide (Bio
Mega/Boehringer-Ingelheim); BM+51.0836: thiazolo-isoindolinone
derivative; BMS 186,318: aminodiol derivative HIV-1 protease
inhibitor (Bristo-Myers-Squibb); d4API:
9-[2,5-dihydro-5-(phosphonomethoxy)-2-furane]adenine (Gilead); d4C:
2',3'-didehydro-2',3'-dideoxycytidine; d4T:
2',3'-didehydro-3'-deoxythymidine (Bristol-Myers-Squibb); ddC;
2',3'-dideoxycytidine (Roche); ddI: 2',3'-dideoxyinosine
(Bristol-Myers-Squibb); DMP-266: 1
1,4-dihydro-2H-3,1-benzoxazin-2-one; DMP-450:
{[4R-(4-a,5-a,6-b,7-b)]-hexahydro-5,6-bis(hydroxy)-1,3-bis(3-amino)phenyl]
methyl)-4,7-bis(phenylmethyl)-2H-1,3-diazepin-2-one}-bismesylate
(Avid); DXG:(-)-.beta.-D-dioxolane-guanosine (Triangle);
EBU-dM:5-ethyl-1-ethoxymethyl-6-(3,5-dimethylbenzyl)uracil; E-EBU:
5-ethyl-1-ethoxymethyl-6-benzyluracil; DS: dextran sulfate;
E-EPSeU: 1-(ethoxymethyl)-(6-phenylselenyl)-5-ethyluracil; E-EPU:
1-(ethoxymethyl)-(6-phenyl-thio)-5-ethyluracil; FTC:
.beta.-.sup.2',3 '-dideoxy-5-fluoro-3'-thiacytidine (Triangle);
HBY097:
S-4-isopropoxycarbonyl-6-methoxy-3-(methylthio-methyl)-3,4-dihydroquinoxal
in-2(1H)-thione; HEPT:
1-[2-hydroxyethoxy)methyl]6-(phenylthio)thymine; HIV-1 :human
immunodeficiency virus type 1; JM2763:
1,1'-(1,3-propanediyl)-bis-1,4,8,11-tetraazacyclotetradecane
(Johnson Matthey); JM3 100:
1,1'-[1,4-phenylenebis-(methylene)]-bis-1,4,8,11-tetraazacyclotetradecane
(Johnson Matthey); KNI-272:
(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid-containing
tripeptide; L-697,593;
5-ethyl-6-methyl-3-(2-phthalimido-ethyl)pyridin-2(1H)-one;
L-735,524: hydroxy-aminopentane amide HIV-1 protease inhibitor
(Merck); L-697,661:
3-{[(-4,7-dichloro-1,3-benzoxazol-2-yl)methyl]amino}-5-ethyl-6-methylpyrid
in-2(1H)-one; L-FDDC: (0)-.beta.-L-5-fluoro-2',3 '-dideoxycytidine;
L-FDOC : (-)-.beta.-L-5-fluoro-dioxolane cytosine; MKC-442:
6-benzyl-1-ethoxymethyl-5-isopropyluracil (I-EBU:
Triangle/Mitsubishi); Nevirapine:
11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyridol[3,2-b:2',3'-e]diazepin-6
-one (Boehringer-Ingelheim); NSC648400:
1-benzyloxymethyl-5-ethyl-6-(alpha-pyridylthio)uracil (E-BPTU);
P9941: [2-pyridylacetyl-IlePheAla-y(CHOH)]2 (Dupont Merck); PFA:
phosphonoformate (foscarnet; Astra); PMEA:
9-(2-phosphonylmethoxyethyl)adenine (Gilead); PMPA:
(R)-9-(2-phosphonylmethoxypropyl)adenine (Gilead); Ro 31-8959:
hydroxyethylamine derivative HIV-l protease inhibitor (Roche);
RPI-312: peptidyl protease inhibitor, 1-[(3s)-.sup.3
-(n-alpha-benzyloxycarbonyl)-1-asparginyl)-amino-2-hydroxy-4-phenylbutyryl
]-n-tert-butyl-1-proline amide; 2720:
6-chloro-3,3-dimethyl-4-(isopropenyloxycarbonyl)-3,4-dihydro-quinoxalin-2(
1H)thione; SC-52151: hydroxyethylurea isostere protease inhibitor
(Searle); SC-55389A: hydroxyethyl-urea isostere protease inhibitor
(Searle); TIBO R82150:
(+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j
k][1,4]-benzodiazepin-2(1H)-thione (Janssen); TIBO 82913:
(+)-(5S)-4,5,6,7,-tetrahydrdo-9-chloro-5-methyl-6-(3-methyl-2-butenyl)imid
azo-[4,5,1jk]-[1,4]benzodiazepin-2(1H)-thione (Janssen); TSAO-m3T:
[2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5'-(4'-amino-1',2'-oxathio
le-2',2'-dioxide)]-b-D-pentofranosyl-N3-methylthymine; U90 152:
1-[3-[1-methylethyl)-amino]-2-pyridinyl]-4-[[5-[(methylsulphonyl)-amino]-1
H-indol-2yl]carbonyl]piperazine; UC: thiocarboxanilide derivatives
(Uniroyal);
UC-781=N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-furancarbot
hioamide;
UC-82=N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]-2-methyl-3-thiophenecar
bothioamide; VB 11,328: hydroxyethyl-sulphonamide protease
inhibitor (Vertex); VS-478:hydroxyethylsulphonamide protease
inhibitor (vertex); XM 323: cyclic urea protease inhibitor (Dupont
Merck).
IV. Ability of 1,3-oxaselenolanyl nucleosides to inhibit the
replication of HIV and HBV
The ability of nucleosides to inhibit HIV can be measured by
various experimental techniques. The technique used herein, and
described in detail below, measures the inhibition of viral
replication in phytohemagglutinin (PHA) stimulated human peripheral
blood mononuclear (PBM) cells infected with HIV-1 (strain LAV). The
amount of virus produced is determined by measuring the virus-coded
reverse transcriptase enzyme. The amount of enzyme produced is
proportional to the amount of virus produced.
EXAMPLE 4
Anti-HIV Activity of 1,3-Oxaselenolanyl Nucleosides
2-Hydroxymethyl-4-(N-5'-cytosin-1'-yl)-1,3-oxaselenolane and
2-hydroxymethyl-4-(N-5'-fluorocytosin-1'-yl)-1,3-oxaselenolane were
tested for anti-HIV activity.
Three-day old phytohemagglutinin-stimulated PBM cells (10.sup.6
cells/ml) from hepatitis B and HIV-1 seronegative healthy donors
were infected with HIV-1 (strain LAV) at a concentration of about
100 times the 50% tissue culture infections dose (TICD 50) per ml
and cultured in the presence and absence of various concentrations
of antiviral compounds.
Approximately one hour after infection, the medium, with the
compound to be tested (2 times the final concentration in medium)
or without compound, was added to the flasks (5 ml; final volume 10
ml). AZT was used as a positive control.
The cells were exposed to the virus (about 2.times.10.sup.5 dpm/ml,
as determined by reverse transcriptase assay) and then placed in a
CO.sub.2 incubator. HIV-1 (strain LAV) was obtained from the Center
for Disease Control, Atlanta, Ga. The methods used for culturing
the PBM cells, harvesting the virus and determining the reverse
transcriptase activity were those described by McDougal, et al. (J.
Immun. Meth. 76, 171-183, 1985) and Spira, et al. (J. Clin. Meth.
25, 97-99, 1987), except that fungizone was not included in the
medium (see Schinazi, et al., Antimicrob. Agents Chemother. 32,
1784-1787 (1988); Id., 34:1061-1067 (1990)).
On day 6, the cells and supernatant were transferred to a 15 ml
tube and centrifuged at about 900 g for 10 minutes. Five ml of
supernatant was removed and the virus was concentrated by
centrifugation at 40,000 rpm for 30 minutes (Beckman 70.1 Ti
rotor). The solubilized virus pellet was processed for
determination of the levels of reverse transcriptase. Results are
expressed in dpm/ml of sampled supernatant. Virus from smaller
volumes of supernatant (1 ml) can also be concentrated by
centriguation prior to solubilization and determination of reverse
transcriptase levels.
The median effective (EC.sub.50) concentration was determined by
the median effect-method (Antimicrob. Agents Chemother. 30, 491-498
(1986)). Briefly, the percent inhibition of virus, as determined
from measurements of reverse transcriptase, is plotted versus the
micromolar concentration of compound. The EC.sub.50 is the
concentration of compound at which there is a 50% inhibition of
viral growth.
Mitogen stimulated uninfected human PBM cells (3.8.times.10.sup.5
cells/ml) were cultured in the presence and absence of drug under
similar conditions as those used for the antiviral assay described
above. The cells were counted after 6 days using a hemacytometer
and the trypan blue exclusion method, as described by Schinazi, et
al., (Antimicrobial Agents and Chemotherapy, 22(3), 499 (1982)).
The IC.sub.50 is the concentration of compound which inhibits 50%
of normal cell growth.
Table 4 provides the EC.sub.50 values (concentration of nucleoside
that inhibits the replication of the virus by 50% in PBM cells,
estimated 10% error factor) and IC.sub.50 values (concentration of
nucleoside that inhibits 50% of the growth of mitogen-stimulated
uninfected human PBM cells, CEM cells, and in Vero cells) of
2-hydroxymethyl-4-(N-5'-cytosin-1'-yl)-1,3-oxaselenolane and
2-hydroxymethyl-4-(N-5'-fluorocytosin-1'-yl)-1,3-oxaselenolane.
TABLE 4 ______________________________________ Anti-HIV activities
of oxaselenolane nucleosides Anti-HIV Toxicity Activity in PBM
(IC.sub.50 .mu.M) PMB Base Cells (EC.sub.50 .mu.M) CEM Vero
______________________________________ Cytosine 0.88 >100 >
100 > 100 5-F-Cytosine 0.07 >100 > 100 > 100
______________________________________
Table 5 provides the percent purity, EC.sub.50 values (.mu.M),
EC.sub.90 values (.mu.M) and IC.sub.50 values in PBM cell of
racemic .beta.-Se-ddC, its (+)- and (-)-isomers and for racemic
.beta.-Se-FddC, its (+)- and (-)-isomers.
TABLE 5 ______________________________________ Anti-HIV Activity
and Cytotoxicity of Racemates and Enantiomers of Oxaselenolane
Cytosine Nucleosides Enanti- % EC.sub.50 EC.sub.90 IC.sub.50
Compound omer Purity .mu.M .mu.M PBM
______________________________________ .beta.-Se-ddc .+-. 50 2.69
209 >100 .beta.-Se-ddC - .apprxeq.100 0.88 5.42 >100
.beta.-Se-ddC + .apprxeq.96 3.39 677 >100 .beta.-Se-FddC .+-. 50
5.55 16.41 >100 .beta.-Se-FddC - .apprxeq.100 0.21 1.05 >100
.beta.-Se-FddC + .apprxeq.100 41.9 164 >100
______________________________________
The anti-HIV activity of .beta.-Se-ddC, its (+)- and (-)-isomers
and racemic .beta.-Se-FddC, its (+)- and (-)-isomers were also
tested in PBM cells infected with HIV that exhibits a mutation at
codon 184 in the reverse transcriptase gene. The results are
provided in Table 6. As indicated, racemic and (-)-.beta.-Se-ddC
exhibits significant activity against the mutated virus.
TABLE 6
__________________________________________________________________________
Effect of Oxaselenolane Cytosine Nucleosides Against Cloned M184
HIV-1 % EC.sub.50 EC.sub.90 FI FI Compound Enantiomer Purity Virus
.mu.M .mu.M EC.sub.50 EC.sub.90
__________________________________________________________________________
.beta.-Se-ddC .+-. 50 xxBRU 1.84 6.90 -- -- .beta.-Se-ddC -
.apprxeq.100 xxBRU 0.11 0.95 -- -- .beta.-Se-ddC + .apprxeq.96
xxBRU 8.62 35.1 -- -- .beta.-Se-FddC .+-. 50 M184V 108 337 59 49
.beta.-Se-FddC - .apprxeq.100 M184V >50 >50 >455 >53
Se-FddC + .apprxeq.96 M184V >50 >50 >6 >1
__________________________________________________________________________
Note: FI (fold increase) EC.sub.50 = EC.sub.50 data from cloned
virus/EC.sub.50 date from xxBRU
EXAMPLE 5
Anti-HBV Activity of 1,3-Oxaselenolanyl Nucleosides
The ability of the active compounds to inhibit the growth of virus
in 2.2.15 cell cultures (HepG2 cells transformed with hepatitis
virion) can be evaluated as described in detail below.
A summary and description of the assay for antiviral effects in
this culture system and the analysis of HBV DNA has been described
(Korba and Milman, 1991, Antiviral Res., 15:217). The antiviral
evaluations are performed on two separate passages of cells. All
wells, in all plates, are seeded at the same density and at the
same time.
Due to the inherent variations in the levels of both intracellular
and extracellular HBV DNA, only depressions greater than 3.5-fold
(for HBV virion DNA) or 3.0-fold (for HBV DNA replication
intermediates) from the average levels for these HBV DNA forms in
untreated cells are considered to be statistically significant
(P<0.05). The levels of integrated HBV DNA in each cellular DNA
preparation (which remain constant on a per cell basis in these
experiments) are used to calculate the levels of intracellular HBV
DNA forms, thereby ensuring that equal amounts of cellular DNA are
compared between separate samples.
Typical values for extracellular HBV virion DNA in untreated cells
range rom 50 to 150 pg/ml culture medium (average of approximately
76 pg/ml). Intracellular HBV DNA replication intermediates in
untreated cells range from 50 to 100 .mu.g/pg cell DNA (average
approximately 74 pg/.mu.g cell DNA). In general, depressions in the
levels of intracellular HBV DNA due to treatment with antiviral
compounds are less pronounced, and occur more slowly, than
depressions in the levels of HBV virion DNA (Korba and milman,
1991, Antiviral Res., 15:217).
The manner in which the hybridization analyses are performed for
these experiments results in an equivalence of approximately 1.0 pg
of intracellular HBV DNA to 2-3 genomic copies per cell and 1.0
pg/ml of extracellular HBV DNA to 3.times.10.sup.5 viral
particles/ml.
Toxicity analyses can be performed to assess whether any observed
antiviral effects are due to a general effect on cell viability.
One method that can be used is the measurement of the uptake of
neutral red dye, a standard and widely used assay for cell
viability in a variety of virus-host systems, including HSV and
HIV. Toxicity analyses are performed in 96-well flat bottomed
tissue culture plates. Cells for the toxicity analyses are cultured
and treated with test compounds with the same schedule as described
for the antiviral evaluations below. Each compound is tested at 4
concentrations, each in triplicate cultures (wells "A", "B", and
"C"). Uptake of neutral red dye is used to determine the relative
level of toxicity. The absorbance of internalized dye at 510 nm
(A.sub.sin) is used for the quantitative analysis. Values are
presented as a percentage of the average A.sub.sin values in 9
separate cultures of untreated cells maintained on the same 96-well
plate as the test compounds.
EXAMPLE 6
Use of 1,3-Oxaselenolanyl Nucleosides to Treat Abnormal Cellular
Proliferation
Some of the 1,3-oxaselenolanyl nucleosides described herein can be
used to treat abnormal cellular proliferation, including tumors and
cancer. The extent of antiproliferative activity can be easily
assessed by assaying the compound according to the procedure below
in a CEM cell or other tumor or proliferative cell line assay. CEM
cells are human lymphoma cells (a T-lymphoblastoid cell line that
can be obtained from ATCC, Rockville, Md.). The toxicity of a
compound to CEM cells provides useful information regarding the
activity of the compound against tumors. The toxicity is measured
as IC.sub.50 micromolar). The IC.sub.50 refers to that
concentration of test compound that inhibits the growth of 50% of
the tumor cells in the culture. The lower the IC.sub.50, the more
active the compound is an antitumor agent. In general, a
1,3-oxaselenolanyl nucleoside exhibits antitumor activity and can
be used in the treatment of abnormal proliferation of cells if it
exhibits a toxicity in CEM or other immortalized tumor cell line of
less than 10 micromolar, more preferably, less than approximately 5
micromolar, and most preferably, less than 1 micromolar.
Drug solutions, including cycloheximide as a positive control, are
plated in triplicate in 50 .mu.l growth medium at 2 times the final
concentration and allowed to equilibrate at 37.degree. C. in a 5%
CO.sub.2 incubator. Log phase cells are added in 50 .mu.l growth
medium to a final concentration of 2.5.times.10.sup.3 (CEM and
SK-MEL-28), 5.times.10.sup.3 (MNAN, MDA-MB-435s, SKMES-1, DU-145,
Lncap), or 1.times.10.sup.4 (PC-3, MCF-7) cells/well and incubated
for 3 (DU-145, PC-3, MNAN), 4 (MCF-7, SK-MEL-28, CEM), or 5
(SK-MES-1, MDA-MB-435s, LNCaP) days at 37.degree. C. under a 5%
CO.sub.2 air atmosphere. Control wells include media alone (blank)
and cells plus media without drug. After growth period, 15 .mu.l of
Cell Titer 96 kit assay dye solution (Promega, Madison, Wis.) are
added to each well and the plates are incubated 8 hr at 37.degree.
C. in a 5% CO.sub.2 incubator. Promega Cell Titer 96 kit assay stop
solution is added to each well and incubated 4-8 hr in the
incubator. Absorbance is read at 570 nm, blanking on the
medium-only wells using a Biotek Biokinetics plate read (Biotek,
Winooski, Vt.). Average percent inhibition of growth compared to
the untreated control is calculated. C.sub.50, IC.sub.90, slope and
r value are calculated by the method of Chou and Talaly. Chou T-C,
Talalay P. Quantitative analysis of dose-effect relationships: The
combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme
Regul. 1984;22:27-55.
IV. Preparation of Pharmaceutical Compositions
Humans suffering from diseases caused by any of the diseases
described herein, including HIVinfection, HBV infection, or
abnormal cellular proliferation, can be treated by administering to
the patient an effective amount of a 1,3-oxaselenolanyl nucleoside
optionally in a pharmaceutically acceptable carrier or diluent. The
active materials can be administered by any appropriate route, for
example, orally, parenterally, intravenously, intradermally,
subcutaneously, or topically, in liquid or solid form.
The active compound is included in the pharmaceutically acceptable
carrier or diluent in an amount sufficient to deliver to a patient
a therapeutically effective amount of compound to inhibit viral
replication in vivo, especially HIV and HBV replication, without
causing serious toxic effects in the patient treated. By
"inhibitory amount" is meant an amount of active ingredient
sufficient to exert an inhibitory effect as measured by, for
example, an assay such as the ones described herein.
A preferred dose of the compound for all the above-mentioned
conditions will be in the range from about 1 to 50 mg/kg,
preferably 1 to 20 mg/kg, of body weight per day, more generally
0.1 to about 100 mg per kilogram body weight of the recipient per
day. The effective dosage range of the pharmaceutically acceptable
derivatives can be calculated based on the weight of the parent
nucleoside to be delivered. If the derivative exhibits activity in
itself, the effective dosage can be estimated as above using the
weight of the derivative, or by other means known to those skilled
in the art.
The compound is conveniently administered in unit or any suitable
dosage form, including but not limited to one containing 7 to 3000
mg, preferably 70 to 1400 mg of active ingredient per unit dosage
form. An oral dosage of 50-1000 mg is usually convenient.
Ideally the active ingredient should be administered to achieve
peak plasma concentrations of the active compound of from about 0.2
to 70 pM, preferably about 1.0 to 10 .mu.M. This may be achieved,
for example, by the intravenous injection of a 0.1 to 5% solution
of the active ingredient, optionally in saline, or administered as
a bolus of the active ingredient.
The concentration of active compound in the drug composition will
depend on absorption, inactivation, and excretion rates of the drug
as well as other factors known to those of skill in the art. It is
to be noted that dosage values will also vary with the severity of
the condition to be alleviated. It is to be further understood that
for any particular subject, specific dosage regimens should be
adjusted over time according to the individual need and the
professional judgment of the person administering or supervising
the administration of the compositions, and that the concentration
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition. The active
ingredient may be administered at once, or may be divided into a
number of smaller doses to be administered at varying intervals of
time.
A preferred mode of administration of the active compound is oral.
Oral compositions will generally include an inert diluent or an
edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition.
The tablets, pills, capsules, troches and the like can contain any
of the following ingredients, or compounds of a similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a disintegrating
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring. When the dosage unit form is a capsule, it can
contain, in addition to material of the above type, a liquid
carrier such as a fatty oil. In addition, dosage unit forms can
contain various other materials which modify the physical form of
the dosage unit, for example, coatings of sugar, shellac, or other
enteric agents.
The compound can be administered as a component of an elixir,
suspension, syrup, wafer, chewing gum or the like. A syrup may
contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
The compound or a pharmaceutically acceptable derivative or salt
thereof can also be mixed with other active materials that do not
impair the desired action, or with materials that supplement the
desired action, such as antibiotics, antifungals,
antiinflammatories, protease inhibitors, or other nucleoside or
nonnucleoside antiviral agents, as discussed in more detail above.
Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; cheating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parental preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
If administered intravenously, preferred carriers are physiological
saline or phosphate buffered saline (PBS).
In a preferred embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation.
Liposomal suspensions (including liposomes targeted to infected
cells with monoclonal antibodies to viral antigens) are also
preferred as pharmaceutically acceptable carriers. These may be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811 (which is
incorporated herein by reference in its entirety). For example,
liposome formulations may be prepared by dissolving appropriate
lipid(s) such as stearoyl phosphatidyl ethanolamine, stearoyl
phosphatidyl choline, arachadoyl phosphatidyl choline, and
cholesterol) in an inorganic solvent that is then evaporated,
leaving behind a thin film of dried lipid on the surface of the
container. An aqueous solution of the active compound or its
monophosphate, diphosphate, and/or triiphosphate derivatives is
then introduced into the container. The container is then swirled
by hand to free lipid material from the sides of the container and
to disperse lipid aggregates, thereby forming the liposomal
suspension.
This invention has been described with reference to its preferred
embodiments. Variations and modifications of the invention, will be
obvious to those skilled in the art from the foregoing detailed
description of the invention. It is intended that all of these
variations and modifications be included within the scope of this
invention.
* * * * *